Quick Estimator 2005 Keygens

Posted By admin On 14.07.19
Published online 2013 Jun 28. doi: 10.2147/IJN.S31678
PMID: 23847416

How could a bought version have a cracked serial number in the first place? Finale 2002b, 2003a, 2004b, 2005b, Win XP SP3, 2011b Win 7 64bit. If you had a cracked version before, the hidden registration files Finale is looking for may have been altered by the crack/keygen software and may not.

This article has been cited by other articles in PMC.

Associated Data

Supplementary Materials
Figure S1: Inhibition of growth of NB4 cells by Ara-C.

Note: Relative viabilities were determined using CCK-8 assays as described in the experimental section and the results were compared with untreated (control) cells.

Abbreviations: Ara-C, cytosine arabinoside; CCK-8, Cell Counting Kit-8.

GUID: 6C96EEF9-CA16-4BC1-AD12-8EA334E28143
Figure S2: Example of FCA of a single cell apoptosis experiment.

Note: Five time points are shown for FCA, 0, 2, 4, 6, and 12 hours after addition of 10−5 M Ara-C; PI+/FITC+ indicates deeply apoptotic or dead cells, while PI−/FITC+ indicates apoptotic cells.

Abbreviations: Ara-C, cytosine arabinoside; FCA, flow cytometric analysis; h, hours.

GUID: 9B0F3AE5-C97C-4928-BAA1-03A6F3D34144
Figure S3: Gene expression profiling using microarray. (A) Gene ontology image of differentially expressed genes. (B) Cluster image of gene expression profiles to Ara-C-induced apoptosis from 0- to 12-hour time points.

Abbreviations: Ara-C, cytosine arabinoside; h, hours.

GUID: F01099D1-5230-4E8F-A337-E4E02F084C7A
Figure S4: Real-time quantitative PCR analysis of the expression of selected genes. The results of real-time quantitative PCR analysis were similar to those microarray gene expression data. (A) Apoptosis-related genes. (B) Cell division- and proliferation-related genes. (C) Microtubule-related genes. (D) Ion transport-related genes.

Abbreviations: PCR, polymerase chain reaction.

GUID: A40A8763-997D-47C0-A397-18AC14D045AB
Figure S5: Systematic transcription regulation pathway map of the apoptosis time course.

Notes: Solid dark lines represent previously known pathways; dashed dark lines represent undefined pathways. Red rectangles represent induced genes with fold changes ≥ 1.5; green rectangles represent repressed genes with fold changes ≥ 0.67. Blue ovals represent hypothetical activated TFs. Further TF ELISA experiments are needed to determine whether p53 or NF-κB activation is involved in Ara-C-induced apoptosis of NB4 cells. All elements are approximately arranged relative to the time axis.

Abbreviations: Ara-C, cytosine arabinoside; NF-κB, nuclear factor κB; TF, transcription factor.

GUID: 403983FE-2225-47BD-9247-12079BED487F
Figure S6: Changes in intracellular calcium, sodium, and potassium over the 12-h time course measured using FCA. (A) An increase in Fluo 4-AM (Ca2+) fluorescence from control cells indicates an increase in intracellular calcium. (B) An increase in SBFI-AM (Na+) fluorescence from control cells indicates an increase in intracellular sodium. (C) A decrease in PBFI-AM (K+) fluorescence from control cells indicates a loss of intracellular potassium.

Notes: The data are shown as histograms which are representative of a single experiment.

Abbreviations: FCA, flow cytometric analysis h, hours.

GUID: 793CE32E-0C1D-4AE6-976C-58B6402DA4DC
Table S1: Sequences of primer pairs used for real-time quantitative PCR
GeneForward primer (5′–3′)Reverse primer (5′–3′)
SESN2AGATGGAGAGCCGCTTTGAGCTCCGAGTGAAGTCCTCATATCCG
E2F2AGCTGGAACCGAGAGAACATGACACGACCAGGCGAAACC
TNFGGCGTGGAGCTGAGAGATAACGGTGTGGGTGAGGAGCACAT
HSPA1AACCTTCGACGTGTCCATCCTGATCCTCCACGAAGTGGTTCACCA
HSPA1BACCTTCGACGTGTCCATCCTGATCCTCCACGAAGTGGTTCACCA
C20orf129GCGTGGCAACAGGCTCCTACAGGCATACAGGATTCGGAACTCC
IRS2CCTGCCCCCTGCCAACACCTTGTGACATCCTGGTGATAAAGCC
DDIT4AGGAAGCTCATTGAGTTGTGGGTACATGCTACACACACAT
KIF20AGTACCAACCAGGAAAATCAGTGTCTGAGTATTGCATCCTG
CENPEGGAGAAAGATGACCTACAGAGGCAGTTCCTCTTCAGTTTCCAGGTG
P2RX4GTGGCGGATTATGTGATACCAGCCACACAGTGGTCGCATCTGGAA
KCTD9GCCGCTGTAATCTTGCACATGCCAGTTTCAGGGATGCTCCTTCTG
GAPDHCGCTCTCTGCTCCTCCTGTTCCATGGTGTCTGAGCGATGT
Table S2: Number of differentially expressed genes
Time (hours)UpDownTotal
2549
4152237
6241842
129522117
Table S3: Total of 152 genes with ≥2.0-fold change at least once for all four time points
Oligo_idGeneRatio
2 hours4 hours6 hours12 hours
H200015775TNF2.27681.61311.88942.1108
H200019847DDIT31.13872.84343.76664.1329
H200011743DDIT41.8655.2085.80265.2344
H200001568SESN21.17822.53412.97562.1333
H200008354TXNIP1.10292.46592.61463.029
H200015433E2F21.34782.09732.33983.3365
H200000027PMAIP11.25041.96173.16813.0868
H200006759GADD45A1.111.70162.16762.1329
H200012407CASP8AP21.46831.79282.12792.1613
H200003690TRIB31.29741.98313.80153.8056
H200006040CHAF1B1.24581.67922.20181.8877
H200005977PRNP1.24091.20691.46492.3403
H200006833NFKBIA1.06241.11481.36962.2215
H200006782IRF11.27291.28681.61452.0059
H200000124ATF31.10521.64941.5592.7846
H200011520NAB11.34861.49771.65882.0771
H200010637E2F81.11421.57731.88932.0674
H200004695REL1.5211.99312.2944
H200015131TNFAIP31.78753.108310.82
H200019156EGR15.9961
H200003705RASSF11.22682.3082
H200003433MOAP12.2819
H200006172ASNS2.1505
H200012696ZNF6243.6537
H200015127PRKCE2.055
H200008824KLF63.2992
H200006354BTG10.84071.17712.17413.1005
H200006664NFIL30.86791.26321.98413.1347
H200005485PAK10.99771.13591.48392.0311
H200008426DUSP60.68070.78261.09392.3816
H200001719HSPA1A0.45330.80860.60380.4123
H200017080HSPA1B0.47460.82060.56590.3964
H200005758EIF4EBP10.56870.35180.31280.4273
H200007392ARHGDIA0.92440.88830.78770.4844
H200001819CCNE11.40232.26792.23771.8367
H200006902TIEG/KLF101.33681.81591.70912.5303
H200000106ADM1.05762.0229
H200000570CXCL1010.3587
H200013414IRS20.46830.79331.1211.0842
H200005718C20orf129/FAM83D0.4910.45940.45460.5191
H200001767TPX20.64550.49210.50830.6906
H200006436DLG7/DLGAP50.7650.48490.51740.445
H200010905BUB10.63020.4480.48730.5465
H200012276ASPM0.78510.44250.50620.6396
H200003347CCNB10.59730.4250.40630.4447
H200005304KNTC2/NDC800.7160.3980.5140.8752
H200006423PLK10.64910.350.334
H200004906CDCA80.76980.5630.49970.6453
H200005764ZNF339/OVOL20.73920.69690.47210.6271
H200006136CENPE0.61140.35470.45070.5201
H200005853KIF20A0.53750.32590.310.411
H200012386ECT20.4648
H200005775KCTD91.74872.74774.1513
H200018987P2RX41.17641.73712.8761
H200003474CLIC41.1811.17571.63142.1938
H200005870CCL32.4173.5841
H200006175CCL43.47273.9945
H200003915TNFAIP611.9615
H2000204902.16932.04091.48411.6363
H200006467HMGCS12.12381.50231.16641.0935
H200005214INSIG12.82371.3731.10141.0765
H200004003CCNE21.66622.66283.83293.4481
H200002187TIPARP1.16512.05552.12952.3472
H200017137CCL3L11.39321.28632.0575.6597
H200006526ATP2B11.1411.09571.60692.9833
H200002418FOXO3A1.12841.10581.56032.667
H200001281HECA1.09331.16251.67182.8152
H2000058341.1051.1771.20492.2256
H200004109RSN1.09811.20931.64782.2426
H200002137KIAA11471.11651.21481.42342.3482
H200002754N4BP21.34141.21471.51332.1562
H200014202RGS161.09031.28471.67353.4525
H200011487CCNB1IP11.19181.29451.52512.2965
H200012358MAP1LC3B1.04031.32941.96772.3889
H2000099571.15461.41681.73512.4842
H200011669C6orf481.13911.57851.47322.7148
H200006727MMD21.7641.68831.85762.657
H200006646CD831.40521.71511.87712.8336
H200002948ATP2B11.16530.96681.29382.2311
H200007999GBP71.19480.95441.08792.2038
H200007904PTPRC1.18870.82260.95542.1509
H200006209C1orf56/MLLT111.27610.930.98832.2463
H200017557OPN31.39181.13230.79390.4675
H2000094441.05651.01790.99520.4823
H200006408MAT2A1.13491.06420.67710.4931
H200000511TBXAS11.09950.90850.74690.4983
H200006231KIAA01821.14520.71090.64250.4688
H200012473PCF111.10020.88680.69820.4149
H2000062601.18050.92380.56780.3931
H200002834FAM111A0.79311.18881.57982.7231
H200008575MEF2A0.99561.08741.22692.1852
H200001006DHRS130.82671.42871.83712.1821
H200017445SUCNR10.85170.80421.22752.6318
H200003843SAT10.90610.97011.10822.316
H200004069PTPRE0.85680.95311.0982.1504
H200006587RGS20.64770.88681.47712.6675
H200008209NAV30.65710.44081.2533.3403
H2000078450.96760.49380.65071.2268
H200021128TCTN10.8480.37280.48921.2922
H200001569RSAD10.97890.94840.85240.4888
H200017562C1GALT1C10.95650.76580.69330.4991
H200002814C9orf1400.93870.79710.72790.4626
H200002245C22orf90.83980.88350.9080.4672
H200020390FAM46A0.80730.70610.65840.4897
H200003471YWHAG0.8520.520.48670.5355
H200000419CENPA0.77220.5420.45330.6846
H200001941FAM46A0.74280.68490.69310.4513
H2000161050.72930.69810.49050.792
H200008360DHRS90.610.48670.63950.7368
H200004769HIST1H4C0.58190.47280.60940.6927
H2000193460.60520.44320.40160.718
H2000187400.59380.43210.52770.7306
H200001067EVI2B0.63040.41670.66910.7823
H200016261STK6/AURKA0.60820.40280.4410.6585
H200013081DEPDC10.55250.38150.4393
H2000201231.06391.24452.0239
H200001749CASP70.95481.41032.544
H200007958CCDC1340.6940.75552.0645
H2000186750.89480.83590.4542
H2000156362.09661.5265
H2000001565.115125.8726126.9222
H200001516PELI11.43421.71912.1665
H2000113630.60850.4937
H200000205HIST2H2AA30.45010.4727
H200015535FICD2.2632.2602
H200016527CXorf34/TRMT2B2.06722.1392
H200005739NEFM1.49992.8711
H200001150ARRDC42.35311.9712
H2000164142.0232
H200017236LIMK21.42112.0302
H200004185CDKN2AIP1.84342.2906
H200003339AVPI11.87192.0933
H200019676LRP112.0051.9775
H200004016PARP162.41332.2154
H200000467SLC18A22.521.9316
H200005857VLDLR3.44393.134
H200004390DMC12.0052
H200012875KIF1B2.5715
H200008545TMEM92.192
H200004439FAM89A2.3003
H200016561PAG12.8081
H200004662PELI22.0424
H200012241TMEM1542.2427
H200009493CYB5R12.5233
H200018222GPR842.3565
H200001460SLC2A32.8812
H200018921OSBPL62.0907
H200006320SDS2.0108
H200002343MASTL2.0644
H200010023CPEB42.2967
H200004544SERPINB82.8708
H200000525PTX33.1971

Abstract

Dielectrophoresis (DEP) can be used to noninvasively measure the dielectric state of the cell, and this data can be used to monitor cell health or apoptosis. In this study, we followed events associated with cytosine arabinoside (Ara-C)-induced apoptosis in NB4 cells using DEP analysis. Our data showed that the membrane capacitance of NB4 cells decreases from 9.42 to 7.63 mF/m2 in the first 2 hours following treatment with Ara-C, and that this decreased capacitance persists for >12 hours. Additionally, cytoplasmic conductivity decreases from 0.217 to 0.190 S/m within 2 hours of Ara-C treatment; this level is maintained for a short period of time before decreasing. We also investigated these events molecularly at the level of gene expression using microarray analysis and showed that the expression of genes related to membrane capacitance and cytoplasmic conductivity change dramatically as early as 2 hours post-Ara-C treatment, and further demonstrated a temporal relationship between the dielectric properties and key events in apoptosis. This study, integrating physical electrical properties of the cell membrane and cytoplasm with those of conductivity-related gene networks, provides new insights into the molecular mechanisms underlying the initiation of apoptosis, establishing a systematic foundation for DEP application in follow-up drug screening and development of medicines for treating leukemia.

Keywords: apoptosis, dielectrophoresis, gene expression profiling, leukemia

Introduction

Approximately 1200 new cases of acute promyelocytic leukemia (APL) are diagnosed in the United States each year (http://ghr.nlm.nih.gov/). The most widely-used anti-metabolite used to induce remission in children and adults with acute leukemia and for APL consolidation therapy to reduce the relapse rate is cytosine arabinoside (Ara-C). Ara-C is a potent killer of dividing cells by inducing cellular apoptosis mechanisms.

Apoptosis is an essential cell process for the ordered destruction of unwanted or damaged cells and can be defined as a gene-directed cellular self-destruction mechanism. Dysfunction of this mechanism is linked to the pathogenesis of cancer and other diseases whereby reduced apoptosis prolongs the persistence of transformed cancer cells. Thus, in vitro technologies for sensitive and rapid assessment of apoptotic events may be useful for understanding cellular events involving the apoptosis mechanism and for aiding disease treatment and anti-cancer drug development.

Many features can be used to distinguish normal cells from cells undergoing apoptosis, such as decreased mitochondrial transmembrane potential, externalization of phosphatidylserine (PS), changes in morphological features, and changes in intracellular ion concentrations. Hence, various methods have been developed to detect apoptosis. The most commonly used method is in vitro measurement of fluorescence probes designed for specific labeling of relevant molecules in apoptotic cells, such as Annexin V and JC-1 assays.– 1 However, these approaches are typically invasive, resulting in cell death.

It has been long recognized that dielectrophoresis (DEP) can be used to determine the dielectric properties of a cell. DEP is noninvasive, does not require markers or labels, and has been developed for a broad range of applications. For instance, DEP can be used for cell characterization, cell isolation, cell separation, drug resistance monitoring, drug assessment, in vitro fertilization, and stem cell research, as well as for analyzing cells in different physiological states, particularly apoptosis.– However, the exact molecular mechanisms that occur during apoptosis remain unclear, as are the events associated with Ara-C-induced apoptosis.

In this study, Ara-C-induced NB4 cell apoptosis was monitored using DEP analysis and validated using standard Annexin V and JC-1 assays. A 22 K human genome oligo array was also used to examine expression levels of genes associated with membrane capacitance and cytoplasmic conductivity in parallel with DEP measurements to comprehensively investigate molecular genetic events that occur during apoptosis.

Material and methods

Cell culture and induction of apoptosis

The NB4 cell line was obtained from Peking Union Medical College. The cells were maintained in RPMI 1640 media (Gibco, Grand Island NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; PAA, Pasching, Austria), and 1% (v/v) penicillin/streptomycin (Invitrogen, Carlsbad CA, USA). The cells were grown at 37°C in a 5% CO2 atmosphere. Ara-C was purchased from Sigma-Aldrich (St Louis, MO, USA). To induce apoptosis, NB4 cells (at a density of 3 × 105 cells/mL) were treated with Ara-C at a final concentration of 1 × 10−5 M. Concentration was determined using CCK-8 assays (see Supplementary material). The incubation period included four time points: 2, 4, 6, and 12 hours.

Detection of apoptosis using Annexin V and JC-1 assays

Apoptosis was analyzed using flow cytometric analysis (FCA). Cells were cultured in 6-well plates, treated with 1 × 10−5 M Ara-C, collected at different time points, and centrifuged at 190 × g for 5 minutes. A rapid Annexin V-FITC apoptosis detection kit (KeyGEN Biotech, Nanjing, China) was used according to the manufacturer’s instructions. Analysis was performed on a BD FACSAria™ II system (BD Biosciences, San Jose, CA, USA). Cells incubated without Ara-C were analyzed in parallel as controls.

JC-1 was used to dye NB4 cells to visually detect early apoptosis. Similarly to the procedure for flow cytometric analysis, cells were collected and incubated in the dark in 0.5 mL incubation buffer (KeyGEN Biotech) with 1 μL JC-1 (2.5 mg/mL) at 37°C for 20 minutes. After incubation, the cells were washed twice with 1 × PBS and analyzed using fluorescence microscopy (DM-IRB; Leica, Wetzlar, Germany). To reduce the presence of dye particulates prior to incubation, the JC-1 solution was sonicated for 5 minutes, followed by centrifugation (1 minute at 9300 × g). The supernatant was used for dye loading.

Dielectrophoretic system setup and crossover frequency determination

The principal components of the DEP system are described in Figure 1. The DEP chip was fabricated by patterning nonclosed ring gold electrodes onto the surface of a clean glass wafer using standard photolithography. As shown in Figure 1A, the width of the electrodes on the glass chip was 20 μm and the inner diameter of the ring was 200 μm. A 200-μL Eppendorf tube was cut and assembled on the chip to construct the DEP measurement chamber (Figure 1B).

Principal components of the DEP assay system. (A) Nonclosed ring gold electrodes, with width of 20 μm and inner diameter of 200 μm. (B) The assembled chip with a reservoir modified from a 200-μL Eppendorf tube. (C) Signal generator. C1: HP33120A, C2: SMB100A. (D) CCD camera coupled to a fluorescence microscope.

Abbreviation: DEP, dielectrophoresis.

The sine signal applied to the chip was generated by a signal generator (HP33120A; Hewlett-Packard Santa Clara, CA, USA) in the range of 10–15 MHz or a signal generator (SMB100A; Rohde and Schwarz, Munich, Germany) in the range of 50–500 MHz at an applied voltage of 3 V (peak-to-peak). Measurements were observed through a CCD camera (DP-71; Olympus, Tokyo, Japan) coupled to a fluorescence microscope (DM-IRB; Leica).

Prior to DEP measurements, the cells were centrifuged at 190 × g for 5 min. The pellets were washed and resuspended in a prepared isotonic medium containing 8.5% (w/v) sucrose and 0.3% (w/v) dextrose buffer for which conductivity had been adjusted to 32.8 mS/m with RPMI 1640 media and a conductivity meter (DDSJ-308A; SPSIC Ltd Shanghai, China). Subsequently, the cells were added to the chip chamber using a micropipette, and the motion of the cells toward or away from the electrode edges due to the applied frequency was observed.

DEP measurements are made by determining crossover frequencies, at which most of the cells in the chamber experience a zero DEP-induced force and exhibit no movement (ie, corresponding to the transition between negative and positive DEP). The crossover frequency for ≥20 cells was measured over a time period of ≤20 minutes for each experiment. The frequency obtained was further used to determine the capacitance of the plasma membrane or cytoplasmic conductivity.

Gene expression profiling and real-time quantitative polymerase chain reaction (PCR)

The 22 K human genomic oligo array (CapitalBio Corp, Beijing, China) contains 21,329 5′-amino-modified 70-mer probes of the Human Genome OligoSet (Version 2.1; Operon, Huntsville, AL, USA). Total RNA was isolated using the Trizol method (Invitrogen). Reverse transcription was performed using M-MLV (Takara Chemicals, Shiga, Japan). Microarray experiments were performed as described previously. After hybridization, microarrays were scanned using a LuxScan™ 10 K/A confocal scanner (CapitalBio), and data from the obtained images were extracted using LuxScan 3.0 software (CapitalBio). Raw data were normalized using the space- and intensity-dependent LOWESS program. For each test and control sample at the 2-, 4-, 6-, and 12-hour time points, hybridization experiments were performed using the dye-swap strategy. Only genes with consistent differential expression (both above a twofold change) in both dye-swap microarrays were selected as differentially expressed genes. The description of this microarray study follows the Minimum Information About a Microarray Experiment (MIAME) guidelines.

A selected subset of differentially expressed genes was validated using real-time quantitative PCR employing an EvaGreen Real-time qPCR Core Reagent Kit (CapitalBio) and a Bio-Rad IQ™ 5 (Bio-Rad, Hercules, CA, USA). Target genes and a reference gene (glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) were amplified in parallel. Primer sequences are shown in Supplementary Table S1. The results were analyzed using Bio-Rad IQ™ 5 (software version 2.1; Bio-Rad). PCR amplification products were analyzed using melting curve analysis and 1.5% agarose gel electrophoresis. The formula EtargetCP1–CP2/EGADPHCP3–CP4 was employed to calculate the relative gene expression ratio of a target gene, where E is the real-time PCR efficiency and CP is the real-time PCR crossing point. All reactions were performed in triplicate.

Detection of morphology changes using scanning electron microscopy (SEM) and indirect immunofluorescence (IF) microscopy

After centrifugation at 190 × g for 5 minutes followed by resuspension, the cells were fixed with 2.5% glutaraldehyde at room temperature for 1 hour, washed in 1 × PBS, and exposed to 1% osmium tetroxide for another hour. The cells were dehydrated by sequential immersion in 30, 50, 70, 80, 90, and 2 × 100% ethanol, followed by a 50/50 ethanol/tert-Butanol solution, and then 100% tert-Butanol. The cells were freeze-dried (ES-2030; Hitachi, Tokyo, Japan) for 4 h, coated with a thin gold layer using an ion sputter (E-1010; Hitachi), and finally attached to the microscope supports using silver glue. The cells were imaged at 15 kV by SEM (Quanta 200; FEI, OR, USA).

For IF analysis, cells were collected washed and plated on glass coverslips. Next, the cells were fixed in 4% paraformaldehyde for 30 min and permeabilized using 0.2% Triton X-100 in 1 × PBS. After blocking with 1% (w/v) bovine serum albumin (BSA) in 1 × PBS, the cells were incubated with rabbit KIF20A polyclonal antibody (Abnova, Taipei, Taiwan) or rabbit y-tubulin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. Subsequently, the cells were washed three times with 1 × PBS and stained with DyLight-labeled goat anti-rabbit IgG (Jackson, West Grove, PA, USA) at room temperature for 4 hours. Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI); Vector Labs, Burlingame, CA, USA). The cells were then examined and photographed using a laser scanning confocal microscope (LSM710; Carl Zeiss, Oberkochen, Germany). The excitation wavelength was 488 nm for DyLight and 405 nm for DAPI. Images were merged using ZEN software (Carl Zeiss).

Measurements of intracellular calcium, sodium, and potassium using flow cytometry

Intracellular calcium ion concentration was measured using Fluo4-AM (Dojindo, Kumamoto, Japan) and 20% pluronic F-127 dissolved in DMSO to make a 1 mM stock solution. Immediately before Fluo 4-AM loading, the cells were gently washed with HEPES-buffered saline (10 mM HEPES, 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, pH 7.35) and then incubated in Fluo 4-AM working solution (5 μM Fluo 4-AM and 0.05% pluronic F-127 dissolved in HEPES buffer) for 30 minutes at 37°C in a cell incubator. Next, the cells were washed with HEPES-buffered saline to remove extracellular Fluo 4-AM and incubated for an additional 20 minutes to allow deesterification of Fluo 4-AM into its active-dye form Fluo 4. Analysis was performed on a BD FACSCalibur™ system (BD Biosciences) at an excitation of 488 nm.

For intracellular sodium and potassium ion measurements, 2 μL of 2.5 mM SBFI-AM (Sigma-Aldrich) and PBFI-AM (Santa Cruz Biotechnology) stock solutions were individually added to 1 mL of cells at a final concentration of 5 μM 1 hour prior to conducting the experiment. Incubation was continued at 37°C in a 5% CO2 atmosphere. Immediately before FCA, propidium iodide (PI; Sigma-Aldrich) was added at a final concentration of 10 μg/mL. Approximately 1 × 104 cells were analyzed by sequential excitation of the cells at 355 nm for SBFI-AM or PBFI-AM and 488 nm for PI, using a BD™ LSR II Flow Cytometer System (BD Biosciences).

Results and discussion

Detecting apoptosis with Annexin V and JC-1 assays

Apoptotic cells display significant externalization of PS, which can be validated using the Annexin V assay. As shown in Supplementary Figure S2, a series of the Annexin V assays based on flow cytometry was performed at different time points. Our results demonstrate that the proportion of Annexin V-positive cells at 2 hours posttreatment was not significantly different compared to control cells, and up to 4 hours posttreatment, the proportion of the positive cells displayed a clear difference (Figure 2A). Among the entire cell population, this proportion increased from 4.51% ± 0.44% at 2 hours to 7.19% ± 0.26% at 4 hours, 12.42% ± 0.68% at 6 hours, and 21.68% + 0.45% at 12 hours.

Detection of apoptosis by Annexin V and JC-1 assays. (A) Percentage of apoptotic cells after different incubation periods with Ara-C as measured using FCA and Annexin V/PI assays. (B) Viable cells display greenish orange fluorescence and apoptotic cells display green fluorescence.

Note: Our results suggest that NB4 cell apoptosis initiated by Ara-C can be detected at 2 h post-treatment using the JC-1 assay.

Abbreviations: Ara-C, cytosine arabinoside; FCA, flow cytometric analysis.

To ensure that apoptosis initiated by Ara-C could be detected as early as possible (ie, at 2 hours posttreatment), assays based on the mitochondria membrane potential (ΔΨm) were performed. A typical early characteristic of apoptosis is permeabilization of the mitochondrial membrane followed by dissipation of ΔΨm, which is measured using the JC-1 assay As shown in Figure 2B, cells displaying greenish orange fluorescence were viable, while cells displaying green fluorescence were apoptotic. After 2 hours of Ara-C treatment, the proportion of apoptotic cells increased. This indicates that NB4 cell apoptosis initiated by Ara-C can be detected as early as 2 hours posttreatment using the JC-1 assay, during which time the mitochondrial permeability transition pore opens and the corresponding ΔΨm is reduced.

DEP analysis during Ara-C-induced apoptosis

DEP analysis is an assay based on measuring changes in dielectric properties (ie, membrane capacitance and cytoplasmic conductivity) that has been applied for monitoring cell apoptosis.– Currently, there are several theoretical models used to extract cell dielectric properties., In this study, we examined the variation in cell dielectric property trends during cell apoptosis. We applied a simple model for measuring crossover frequencies in an isotonic medium with a specific conductivity (32.8 mS/m)., DEP analysis reveals two types of crossover frequencies, the lower and upper crossover frequencies, which are indicated by ƒx1 and ƒx2, respectively. ƒx1 provides a direct measurement of membrane capacitance, while fx2 provides a direct measurement of cytoplasmic conductivity. As shown in Figure 3A and ​andB,B, crossover frequency of the cells at 2 h post-exposure to Ara-C displayed distinct differences with that obtained from control cells; moreover, ƒx1 continually increased from 96 ± 4.73 to 354 ± 6.11 kHz, while fx2 decreased from 301 ± 7.09 to 165 ± 7.78 MHz over the 12-hour time course. Additionally, cell diameter decreased from 16.30 ± 0.35 to 13.81± 0.62 μm (Figure 3C). As shown in Table 1, membrane capacitance (Cm) decreased from 9.42 ± 0.47 to 3.03 ± 0.05 mF/m2 over the 12-hour time course based on the formula of Pethig and Talary (Cm=2/2πrfX1σs), where r and σs represent the cell radius and conductivity of the surrounding solution (32.8 mS/m), respectively. The decreasing trend of membrane capacitance observed in our experiments is consistent with the observations of previous studies., Similarly, we calculated that cytoplasmic conductivity (σ) decreased from 0.217 ± 0.005 to 0.120 ± 0.006 S/m over the 12-hour time course based on the equation (σcyto=22πεsfX2), where ɛs indicates the relative permittivity of the surrounding solution is a constant, about 81. This generally agrees with previous studies examining Jurkat T-cells undergoing etoposide-induced apoptosis. Compared to the Annexin V and JC-1 assays, the DEP assay can be used to monitor apoptosis noninvasively without the use of added markers or labels.

Monitoring of Ara-C induced apoptosis by DEP analysis. (A and B) Distinct differences in crossover frequency occur between control and the apoptotic NB4 cells as early as 2 h post-exposure to Ara-C. ƒx1 increased from 96 ± 4.73 to 354 ± 6.11 KHz, while ƒx2 decreased from 301 ± 7.09 to 165 ± 7.78 MHz over the 12-h time course. (C) Cell diameter decreased from 16.30 ± 0.35 to 13.81 ± 0.62 μm in apoptotic cells.

Note: No significant change was observed for control cells.

Abbreviations: Ara-C, cytosine arabinoside; DEP, dielectrophoresis.

Table 1

Changes in membrane capacitance (Cm) or cytoplasmic conductivity (σcyto) during Ara-C-induced apoptosis

Time (hours)Cm(mF/m2)σcyto (S/m)
0 (control)9.42 ± 0.470.217 ± 0.005
27.63 ± 0.250.190 ± 0.005
45.75 ± 0.210.188 ± 0.004
64.93 ± 0.200.160 ± 0.004
123.03 ± 0.050.120 ± 0.006

Gene expression profiling during Ara-C-induced apoptosis

To better understand the molecular mechanism underlying apoptosis using DEP monitoring, we examined mRNA changes in NB4 cells after Ara-C treatment over a 12-hour time course using a human whole genome oligo array. A total of 9, 37, 42, and 117 genes displayed altered expression levels at 2, 4, 6, and 12 hours, respectively, as shown in Supplementary Table S2. A total of 152 genes showing ≥2.0-fold change at least once for all four time points (Supplementary Table S3) were selected for gene ontology (GO) analysis and cluster analysis (Supplementary Figure S3).

GO analysis revealed that differentially expressed genes can be grouped into five main categories: apoptosis, cell division and proliferation, cell morphogenesis, ion transport and cell polarity, and others. Additionally, cluster analysis confirmed the expression level of some known apoptosis-related genes changed after Ara-C treatment, including the up-regulated pro-apoptosis genes SESN2, E2F2, DDIT4, TNF, GADD45A, and PMAIPI and the downregulated antiapoptosis genes HSPA1A, HSPA1B, and ARHGDIA (Figure 4, Supplementary Figure S4(A)). In contrast, the expression level of nearly all genes involved in cell division and proliferation was downregulated (eg, C20orf129 and IRS2) (Figure 4, Supplementary Figure S4(B)). Notably, the expression level of some genes involved in cell morphogenesis or ion transport changed as early as 2 hours post-Ara-C treatment, which may correlate with the measured alteration in membrane capacitance or alteration in cytoplasmic conductivity (Figure 4, Supplementary Figure S4(C and D)). Furthermore, we analyzed expression changes in terms of transcriptional regulation of defined gene networks and pathways and present a model of the dynamic and temporal network of pathway activities related to Ara-C-induced apoptosis (Supplementary Figure S5), which may have implications for identifying potential new effectors useful for treating APL.

Relationship of molecular events to the time course of Ara-C-induced apoptosis.

Note: Cluster image of gene expression data of the four main categories: apoptosis, cell division and proliferation, cell morphogenesis, and ion transport and cell polarity.

Abbreviation: Ara-C, cytosine arabinoside.

The original data from this study are available at the Gene Expression Omnibus (see http://www.ncbi.nlm.nih.gov/geo); the accession number is GSE32992. Results of real-time quantitative PCR analysis for all selected genes were in agreement with microarray data (Supplementary Figure S4).

Systematic analysis of changes in dielectric properties

Because DEP analysis is noninvasive, nondestructive, and rapid it has been widely employed for cell apoptosis studies.– However, little is known regarding the temporal relationship between dielectric properties measured using DEP analysis and key events in apoptosis. Here, we used DEP analysis to systematically investigate potential mechanisms responsible at the gene level and at the cellular level for changes in membrane capacitance and cytoplasmic conductivity occurring during apoptosis.

As described above, membrane capacitance decreased from 9.42 ± 0.47 to 3.03 ± 0.05 mF/m2 over 12 hours. Cell membrane capacitance largely depends on plasma membrane surface morphology, such as microvilli, ruffles, folds, and blebs;– cells treated with Ara-C, as well as control cells, were imaged using SEM (Figure 5A). Control cells were covered with numerous microvilli, which appeared to be homogeneous both in size (~16 μm diameter) and surface morphology. However, treated cells exhibited alterations in their plasma membrane morphology. After 2 hours of treatment with Ara-C, the number of microvilli slightly decreased, and the membrane surface became smoother. After a treatment time of ≥4 hours, cell shrinkage and bleb formation were observed. Membrane surface morphology is closely associated with the cytoskeleton and thus can be monitored by observing changes in microtubule-related proteins during apoptosis., As shown in Figure 5B, the average cell fluorescence intensity (FI) related to γ-tubulin from the IF assay gradually decreased over the 12-hour time course, indicating that in cells treated with Ara-C, micro-tubules were disrupted and collapsed, resulting in obvious changes to the cytoskeleton as observed by others. Changes in surface morphology involving the cytoskeleton effectively decreased the surface area of the cell membrane and, hence, decreased cell membrane capacitance.

Changes in morphological features of cells over the 12-h time course of Ara-C induced apoptosis. (A) SEM images of NB4 cells after treatment for 0 (control), 2, 4, 6, and 12 h with Ara-C. The cells displayed progressive changes in membrane morphology as apoptosis developed. (B and C) Merged confocal scanning laser microscopy images represent the changes in microtubule-related proteins over the 12-h time course: (B) γ-tubulin and (C) KIF20A.

Quick estimator 2005 keygen

Note: Cell nuclei are stained with DAPI (blue).

Abbreviations: Ara-C, cytosine arabinoside, SEM, scanning electron microscopy; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride.

We examined the expression level of microtubule-related genes, such as kinesin family member 20A (KIF20A) and centromere-associated protein E (CENPE), and found that these genes were downregulated over the 12-hour time course. More precisely, expression of KIF20A and CENPE decreased to a minimum at 4 hours posttreatment and then remained low for the remainder of the time course (Supplementary Figure S4(C)). Accordingly, the decline in average cell fluorescence intensity related to KIF20A in the IF assay reflects the decreasing KIF20A protein quantity and activity (Figure 5C), which was similar to γ-tubulin. Because of the close association between membrane surface morphology and KIF20A, a cytoskeletal component, the decline in KIF20A expression may induce structural changes to the cell membrane (Figure 5A), further decreasing cell membrane capacitance., Subsequently, an increase in ƒx1 was observed as a consequence of these changes.

Additionally, cytoplasmic conductivity decreased from 0.217 ± 0.005 to 0.120 ± 0.006 S/m. σcyto is another important cell dielectric property which is, to a large extent, determined by ion mobility and concentration in the cytoplasm. Our gene expression profiling results demonstrated that expression of ion transport-related genes, such as P2X purinoceptor 4 (P2RX4; associated with calcium and sodium transport), and potassium channel tetramerisation domain containing 9 (KCTD9; associated with potassium transport), was up-regulated over the 12-hour time course (Supplementary Figure S4(D)). Moreover, changes in intracellular calcium, sodium, and potassium concentration were measured using fluorescence probe-based flow cytometry, and all changes in cytoplasmic conductivity over the 12-hour time course were estimated using the equation (k = F ∑ ZiCiUi) of Bard and Faulkner.47 The experimental data revealed an increase in intracellular calcium and sodium, as well as a loss of intracellular potassium, during apoptosis (Supplementary Figure S6(A–C)). Figure 6 shows the relative dynamic and temporal changes in intracellular ions and cytoplasmic conductivity over the 12-hour time course. Intracellular calcium and sodium increased to >1.5 and 1.2 times their normal levels, respectively, while intracellular potassium level was reduced to <0.80 times its normal level over the 12-hour time course (Figure 6A). A decrease in intracellular potassium ultimately induces a loss of cytoplasmic conductivity because it is a highly abundant intracellular ion,48 even when intracellular calcium and sodium levels increase. Estimated relative changes of average cytoplasmic conductivity based on ion levels decreased significantly at 2 hours post-Ara-C treatment and changed minimally thereafter. T DEP analysis showed nearly the same results over the 6-hour time course. However, the difference became significant at the 12-hour time point (Figure 6B). One possible reason for this difference may be that the ions contributing to cytoplasmic conductivity were limited. Based on gene expression profiling, we found that the expression level of chloride intracellular channel 4 (CLIC4; associated with chloride transport), was significantly up-regulated at the 12-hour time point (Supplementary Table S3), indicating that an apparent change of intracellular chloride ions may have occurred, further affecting overall cytoplasmic conductivity. As a label-free method for measuring overall cytoplasmic conductivity, DEP analysis may be a better choice as a simple and quick monitoring method.

Relative dynamic and temporal changes in intracellular ion levels and cytoplasmic conductivity over the 12-h time course. (A) Fold changes in intracellular calcium, sodium, and potassium from the 0- to 12-hour time points during Ara-C-induced apoptosis. The data represent averages of three independent experiments. (B) Estimated changes in average cytoplasmic conductivity based on changes in intracellular ions levels (blue) or DEP analysis (red) over the 12-h time course.

Abbreviations: Ara-C, cytosine arabinoside; DEP, dielectrophoresis.

Conclusion

In this study, we used DEP analysis to investigate the mechanisms responsible for apoptosis from the gene and cell levels by performing a series of biological assays (gene expression profiling, SEM, IF, and FCA). At the gene level, our studies indicate that expression levels of KIF20A and CENPE, which are related to membrane capacitance, are down-regulated, while expression levels of P2RX4 and KCTD9, which are related to cytoplasmic conductivity, are up-regulated as early as 2 hours post-Ara-C treatment. Accordingly, at the cellular level, the FI related to γ-tubulin and KIF20A gradually decreased, and obvious structural changes to the cell membrane were observed, leading to the loss of membrane capacitance by decreasing the effective surface area of the cell membrane. Additionally, a decrease in intracellular potassium concentration induces a loss of cytoplasmic conductivity, even though the intracellular calcium and sodium levels increase. All of these changes in cell membrane capacitance and cytoplasmic conductivity make apoptotic cells amenable for characterization using DEP analysis, which may be used to help physicians detect apoptosis earlier. As this technology develops, individually tailored and more personalized patient treatment will be possible.

Supplementary material

Methods

Determining Ara-C concentration The appropriate working concentration of Ara-C was evaluated using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assays. Cells were plated at a density of 1 × 105 cells/mL in a 96-well plate. Incubation times and concentrations of Ara-C are shown with the results. After incubation, cell viability was determined using CCK-8 according to the manufacturer’s protocol. Absorbance at 450 and 630 nm was read using a Microplate Reader (Bio-Rad). The experiment was repeated in triplicate.

Results and discussion

As shown in Supplementary Figure S1, several different concentrations were used to test the effect of Ara-C on cell viability at various time points. IC50 (defined as the concentration of drug that reduces cell viability by 50% relative to untreated controls) values were calculated to be approximately 10−4 or 10−5 M for 12 h. Combined with the results of a previous report, a concentration of 10−5 M Ara-C was selected to induce cell apoptosis.

Figure S1

Inhibition of growth of NB4 cells by Ara-C.

Note: Relative viabilities were determined using CCK-8 assays as described in the experimental section and the results were compared with untreated (control) cells.

Abbreviations: Ara-C, cytosine arabinoside; CCK-8, Cell Counting Kit-8.

Figure S2

Example of FCA of a single cell apoptosis experiment.

Note: Five time points are shown for FCA, 0, 2, 4, 6, and 12 hours after addition of 10−5 M Ara-C; PI+/FITC+ indicates deeply apoptotic or dead cells, while PI−/FITC+ indicates apoptotic cells.

Abbreviations: Ara-C, cytosine arabinoside; FCA, flow cytometric analysis; h, hours.

Figure S3

Gene expression profiling using microarray. (A) Gene ontology image of differentially expressed genes. (B) Cluster image of gene expression profiles to Ara-C-induced apoptosis from 0- to 12-hour time points.

Abbreviations: Ara-C, cytosine arabinoside; h, hours.

Quick Estimator 2005 Keygen

Figure S4

Real-time quantitative PCR analysis of the expression of selected genes. The results of real-time quantitative PCR analysis were similar to those microarray gene expression data. (A) Apoptosis-related genes. (B) Cell division- and proliferation-related genes. (C) Microtubule-related genes. (D) Ion transport-related genes.

Abbreviations:Text presets after effects free download. PCR, polymerase chain reaction.

Figure S5

Systematic transcription regulation pathway map of the apoptosis time course.

Notes: Solid dark lines represent previously known pathways; dashed dark lines represent undefined pathways. Red rectangles represent induced genes with fold changes ≥ 1.5; green rectangles represent repressed genes with fold changes ≥ 0.67. Blue ovals represent hypothetical activated TFs. Further TF ELISA experiments are needed to determine whether p53 or NF-κB activation is involved in Ara-C-induced apoptosis of NB4 cells. All elements are approximately arranged relative to the time axis.

Abbreviations: Ara-C, cytosine arabinoside; NF-κB, nuclear factor κB; TF, transcription factor.

Figure S6

Changes in intracellular calcium, sodium, and potassium over the 12-h time course measured using FCA. (A) An increase in Fluo 4-AM (Ca2+) fluorescence from control cells indicates an increase in intracellular calcium. (B) An increase in SBFI-AM (Na+) fluorescence from control cells indicates an increase in intracellular sodium. (C) A decrease in PBFI-AM (K+) fluorescence from control cells indicates a loss of intracellular potassium.

Notes: The data are shown as histograms which are representative of a single experiment.

Abbreviations: FCA, flow cytometric analysis h, hours.

Table S1

Sequences of primer pairs used for real-time quantitative PCR

GeneForward primer (5′–3′)Reverse primer (5′–3′)
SESN2AGATGGAGAGCCGCTTTGAGCTCCGAGTGAAGTCCTCATATCCG
E2F2AGCTGGAACCGAGAGAACATGACACGACCAGGCGAAACC
TNFGGCGTGGAGCTGAGAGATAACGGTGTGGGTGAGGAGCACAT
HSPA1AACCTTCGACGTGTCCATCCTGATCCTCCACGAAGTGGTTCACCA
HSPA1BACCTTCGACGTGTCCATCCTGATCCTCCACGAAGTGGTTCACCA
C20orf129GCGTGGCAACAGGCTCCTACAGGCATACAGGATTCGGAACTCC
IRS2CCTGCCCCCTGCCAACACCTTGTGACATCCTGGTGATAAAGCC
DDIT4AGGAAGCTCATTGAGTTGTGGGTACATGCTACACACACAT
KIF20AGTACCAACCAGGAAAATCAGTGTCTGAGTATTGCATCCTG
CENPEGGAGAAAGATGACCTACAGAGGCAGTTCCTCTTCAGTTTCCAGGTG
P2RX4GTGGCGGATTATGTGATACCAGCCACACAGTGGTCGCATCTGGAA
KCTD9GCCGCTGTAATCTTGCACATGCCAGTTTCAGGGATGCTCCTTCTG
GAPDHCGCTCTCTGCTCCTCCTGTTCCATGGTGTCTGAGCGATGT

Table S2

Number of differentially expressed genes

Time (hours)UpDownTotal
2549
4152237
6241842
129522117

Table S3

Total of 152 genes with ≥2.0-fold change at least once for all four time points

Oligo_idGeneRatio
2 hours4 hours6 hours12 hours
H200015775TNF2.27681.61311.88942.1108
H200019847DDIT31.13872.84343.76664.1329
H200011743DDIT41.8655.2085.80265.2344
H200001568SESN21.17822.53412.97562.1333
H200008354TXNIP1.10292.46592.61463.029
H200015433E2F21.34782.09732.33983.3365
H200000027PMAIP11.25041.96173.16813.0868
H200006759GADD45A1.111.70162.16762.1329
H200012407CASP8AP21.46831.79282.12792.1613
H200003690TRIB31.29741.98313.80153.8056
H200006040CHAF1B1.24581.67922.20181.8877
H200005977PRNP1.24091.20691.46492.3403
H200006833NFKBIA1.06241.11481.36962.2215
H200006782IRF11.27291.28681.61452.0059
H200000124ATF31.10521.64941.5592.7846
H200011520NAB11.34861.49771.65882.0771
H200010637E2F81.11421.57731.88932.0674
H200004695REL1.5211.99312.2944
H200015131TNFAIP31.78753.108310.82
H200019156EGR15.9961
H200003705RASSF11.22682.3082
H200003433MOAP12.2819
H200006172ASNS2.1505
H200012696ZNF6243.6537
H200015127PRKCE2.055
H200008824KLF63.2992
H200006354BTG10.84071.17712.17413.1005
H200006664NFIL30.86791.26321.98413.1347
H200005485PAK10.99771.13591.48392.0311
H200008426DUSP60.68070.78261.09392.3816
H200001719HSPA1A0.45330.80860.60380.4123
H200017080HSPA1B0.47460.82060.56590.3964
H200005758EIF4EBP10.56870.35180.31280.4273
H200007392ARHGDIA0.92440.88830.78770.4844
H200001819CCNE11.40232.26792.23771.8367
H200006902TIEG/KLF101.33681.81591.70912.5303
H200000106ADM1.05762.0229
H200000570CXCL1010.3587
H200013414IRS20.46830.79331.1211.0842
H200005718C20orf129/FAM83D0.4910.45940.45460.5191
H200001767TPX20.64550.49210.50830.6906
H200006436DLG7/DLGAP50.7650.48490.51740.445
H200010905BUB10.63020.4480.48730.5465
H200012276ASPM0.78510.44250.50620.6396
H200003347CCNB10.59730.4250.40630.4447
H200005304KNTC2/NDC800.7160.3980.5140.8752
H200006423PLK10.64910.350.334
H200004906CDCA80.76980.5630.49970.6453
H200005764ZNF339/OVOL20.73920.69690.47210.6271
H200006136CENPE0.61140.35470.45070.5201
H200005853KIF20A0.53750.32590.310.411
H200012386ECT20.4648
H200005775KCTD91.74872.74774.1513
H200018987P2RX41.17641.73712.8761
H200003474CLIC41.1811.17571.63142.1938
H200005870CCL32.4173.5841
H200006175CCL43.47273.9945
H200003915TNFAIP611.9615
H2000204902.16932.04091.48411.6363
H200006467HMGCS12.12381.50231.16641.0935
H200005214INSIG12.82371.3731.10141.0765
H200004003CCNE21.66622.66283.83293.4481
H200002187TIPARP1.16512.05552.12952.3472
H200017137CCL3L11.39321.28632.0575.6597
H200006526ATP2B11.1411.09571.60692.9833
H200002418FOXO3A1.12841.10581.56032.667
H200001281HECA1.09331.16251.67182.8152
H2000058341.1051.1771.20492.2256
H200004109RSN1.09811.20931.64782.2426
H200002137KIAA11471.11651.21481.42342.3482
H200002754N4BP21.34141.21471.51332.1562
H200014202RGS161.09031.28471.67353.4525
H200011487CCNB1IP11.19181.29451.52512.2965
H200012358MAP1LC3B1.04031.32941.96772.3889
H2000099571.15461.41681.73512.4842
H200011669C6orf481.13911.57851.47322.7148
H200006727MMD21.7641.68831.85762.657
H200006646CD831.40521.71511.87712.8336
H200002948ATP2B11.16530.96681.29382.2311
H200007999GBP71.19480.95441.08792.2038
H200007904PTPRC1.18870.82260.95542.1509
H200006209C1orf56/MLLT111.27610.930.98832.2463
H200017557OPN31.39181.13230.79390.4675
H2000094441.05651.01790.99520.4823
H200006408MAT2A1.13491.06420.67710.4931
H200000511TBXAS11.09950.90850.74690.4983
H200006231KIAA01821.14520.71090.64250.4688
H200012473PCF111.10020.88680.69820.4149
H2000062601.18050.92380.56780.3931
H200002834FAM111A0.79311.18881.57982.7231
H200008575MEF2A0.99561.08741.22692.1852
H200001006DHRS130.82671.42871.83712.1821
H200017445SUCNR10.85170.80421.22752.6318
H200003843SAT10.90610.97011.10822.316
H200004069PTPRE0.85680.95311.0982.1504
H200006587RGS20.64770.88681.47712.6675
H200008209NAV30.65710.44081.2533.3403
H2000078450.96760.49380.65071.2268
H200021128TCTN10.8480.37280.48921.2922
H200001569RSAD10.97890.94840.85240.4888
H200017562C1GALT1C10.95650.76580.69330.4991
H200002814C9orf1400.93870.79710.72790.4626
H200002245C22orf90.83980.88350.9080.4672
H200020390FAM46A0.80730.70610.65840.4897
H200003471YWHAG0.8520.520.48670.5355
H200000419CENPA0.77220.5420.45330.6846
H200001941FAM46A0.74280.68490.69310.4513
H2000161050.72930.69810.49050.792
H200008360DHRS90.610.48670.63950.7368
H200004769HIST1H4C0.58190.47280.60940.6927
H2000193460.60520.44320.40160.718
H2000187400.59380.43210.52770.7306
H200001067EVI2B0.63040.41670.66910.7823
H200016261STK6/AURKA0.60820.40280.4410.6585
H200013081DEPDC10.55250.38150.4393
H2000201231.06391.24452.0239
H200001749CASP70.95481.41032.544
H200007958CCDC1340.6940.75552.0645
H2000186750.89480.83590.4542
H2000156362.09661.5265
H2000001565.115125.8726126.9222
H200001516PELI11.43421.71912.1665
H2000113630.60850.4937
H200000205HIST2H2AA30.45010.4727
H200015535FICD2.2632.2602
H200016527CXorf34/TRMT2B2.06722.1392
H200005739NEFM1.49992.8711
H200001150ARRDC42.35311.9712
H2000164142.0232
H200017236LIMK21.42112.0302
H200004185CDKN2AIP1.84342.2906
H200003339AVPI11.87192.0933
H200019676LRP112.0051.9775
H200004016PARP162.41332.2154
H200000467SLC18A22.521.9316
H200005857VLDLR3.44393.134
H200004390DMC12.0052
H200012875KIF1B2.5715
H200008545TMEM92.192
H200004439FAM89A2.3003
H200016561PAG12.8081
H200004662PELI22.0424
H200012241TMEM1542.2427
H200009493CYB5R12.5233
H200018222GPR842.3565
H200001460SLC2A32.8812
H200018921OSBPL62.0907
H200006320SDS2.0108
H200002343MASTL2.0644
H200010023CPEB42.2967
H200004544SERPINB82.8708
H200000525PTX33.1971

Acknowledgments

This work was supported by the grant of National HighTech Program of China (No 2006AA020701). We are grateful to Dr Caiming Xu of Peking Union Medical College for providing the NB4 cell line. We thank Rui Ma and Chao Han for helping with the dielectrophoretic system setup. We also thank Dr Lan Xie and Dr Lei Wang for their helpful discussions and critical reading of the manuscript.

Footnotes

Disclosure

The authors have no conflicts of interest to disclose.

References

1. Grant S. Ara-C: Cellular and molecular pharmacology. Adv Cancer Res. 1998;72:197–233. [PubMed] [Google Scholar]
2. Schlenk RF, Germing U, Hartmann F, et al. High-dose cytarabine and mitoxantrone in consolidation therapy for acute promyelocytic leukemia. Leukemia. 2005;19(6):978–983. [PubMed] [Google Scholar]
3. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science. 1995;267(5203):1456–1462. [PubMed] [Google Scholar]
4. Ly JD, Grubb DR, Lawen A. The mitochondrial membrane potential (ΔΨm) in apoptosis; an update. Apoptosis. 2003;8(2):115–128. [PubMed] [Google Scholar]
5. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol. 1992;148(7):2207–2216. [PubMed] [Google Scholar]
6. van Engeland M, Kuijpers HJH, Ramaekers FCS, Reutelingsperger CPM, Schutte B. Plasma membrane alterations and cytoskeletal changes in apoptosis. Exp Cell Res. 1997;235(2):421–430. [PubMed] [Google Scholar]
7. Wyllie AH. Apoptosis (The 1992 Rose, Frank Memorial Lecture) Br J Cancer. 1993;67(2):205–208.[PMC free article] [PubMed] [Google Scholar]
8. Bortner CD, Cidlowski JA. Uncoupling cell shrinkage from apoptosis reveals that Na+ influx is required for volume loss during programmed cell death. J Biol Chem. 2003;278(40):39176–39184. [PubMed] [Google Scholar]
9. Rodriguez M, Schaper J. Apoptosis: measurement and technical issues. J Mol Cell Cardiol. 2005;38(1):15–20. [PubMed] [Google Scholar]
10. Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨm changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 1997;411(1):77–82. [PubMed] [Google Scholar]
11. Vermes I, Haanen C, Steffensnakken H, Reutelingsperger CJ. A novel assay for apoptosis flow cytometric detection of phosphatidylserine early apoptotic cells using fluorescein labeled expression on Annexin V. J Immunol Methods. 1995;184(1):39–51. [PubMed] [Google Scholar]
12. Sanchis A, Brown AP, Sancho M, et al. Dielectric characterization of bacterial cells using dielectrophoresis. Bioelectromagnetics. 2007;28(5):393–401. [PubMed] [Google Scholar]
13. Gascoyne PRC, Noshari J, Anderson TJ, Becker FF. Isolation of rare cells from cell mixtures by dielectrophoresis. Electrophoresis. 2009;30(8):1388–1398.[PMC free article] [PubMed] [Google Scholar]
14. Wang XB, Yang J, Huang Y, Vykoukal J, Becker FF, Gascoyne PRC. Cell separation by dielectrophoretic field-flow-fractionation. Anal Chem. 2000;72(4):832–839.[PMC free article] [PubMed] [Google Scholar]
15. Labeed FH, Coley HM, Thomas H, Hughes MP. Assessment of multidrug resistance reversal using dielectrophoresis and flow cytometry. Biophys J. 2003;85(3):2028–2034.[PMC free article] [PubMed] [Google Scholar]
16. Hoettges KF, Hübner Y, Broche LM, Ogin SL, Kass GEN, Hughes MP. Dielectrophoresis-activated multiwell plate for label-free high-throughput drug assessment. Anal Chem. 2008;80(6):2063–2068. [PubMed] [Google Scholar]
17. Valley JK, Swinton P, Boscardin WJ, et al. Preimplantation mouse embryo selection guided by light-induced dielectrophoresis. PLoS ONE. 2010;5(4):e10160.[PMC free article] [PubMed] [Google Scholar]
18. Flanagan LA, Lu J, Wang L, et al. Unique dielectric properties distinguish stem cells and their differentiated progeny. Stem Cells. 2008;26(3):656–665. [PubMed] [Google Scholar]
19. Vykoukal J, Vykoukal DM, Freyberg S, Alt EU, Gascoyne PRC. Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation. Lab Chip. 2008;8(8):1386–1393.[PMC free article] [PubMed] [Google Scholar]
20. Chin S, Hughes MP, Coley HM, Labeed FH. Rapid assessment of early biophysical changes in K562 Cells during apoptosis determined using dielectrophoresis. Int J Nanomedicine. 2006;1(3):333–337.[PMC free article] [PubMed] [Google Scholar]
21. Labeed FH, Hughes MP, Thomas H, Coley HM. Rapid assay of apoptotic progress of mixed populations by dielectrophoresis; Proceedings of the American Association for Cancer Research Annual Meeting; Orlando, FL, USA. March 27–31, 2004. [Google Scholar]
22. Labeed FH, Coley HM, Hughes MP. Differences in the biophysical properties of membrane and cytoplasm of apoptotic cells revealed using dielectrophoresis. Biochim Biophys Acta. 2006;1760(6):922–929. [PubMed] [Google Scholar]
23. Pethig R, Talary MS. Dielectrophoretic detection of membrane morphology changes in Jurkat T-cells undergoing etoposide-induced apoptosis. IET Nanobiotechnol. 2007;1(1):2–9. [PubMed] [Google Scholar]
24. Wang XJ, Becker FF, Gascoyne PRC. Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation. Biochim Biophys Acta. 2002;1564(2):412–420.[PMC free article] [PubMed] [Google Scholar]
25. Broche LM, Labeed FH, Hughes MP. Extraction of dielectric properties of multiple populations from dielectrophoretic collection spectrum data. Phys Med Biol. 2005;50(10):2267–2274. [PubMed] [Google Scholar]
26. Yu J, Zhang L, Chen AL, et al. Identification of the gene transcription and apoptosis mediated by TGF-beta-Smad2/3-Smad4 signaling. J Cell Physiol. 2005;215(2):422–433. [PubMed] [Google Scholar]
27. Yang YH, Dudoit S, Luu P, et al. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002;30(4):1–10.[PMC free article] [PubMed] [Google Scholar]
28. Brazma A, Hingamp P, Quackenbush J, et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001;29(4):365–371. [PubMed] [Google Scholar]
29. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29(9):e45.[PMC free article] [PubMed] [Google Scholar]
30. Lei U, Sun PH, Pethig R. Refinement of the theory for extracting cell dielectric properties from dielectrophoresis and electrorotation experiments. Biomicrofluidics. 2011;5(4):044109.[PMC free article] [PubMed] [Google Scholar]
31. Lei U, Lo YJ. Review of the theory of generalised dielectrophoresis. IET Nanobiotechnol. 2011;5(3):86–106. [PubMed] [Google Scholar]
Keygens
32. Arnold WM, Klarmann BG, Sukhorukov VL, Zimmermann U. Membrane accommodation in hypo-osmotically-treated, and in giant electrofused cells. Biochem Soc Trans. 1992;20(2):120S. [PubMed] [Google Scholar]
33. Gascoyne PRC, Vykoukal JV. Dielectrophoresis-based sample handling in general-purpose programmable diagnostic instruments. Proc IEEE Inst Electr Electron Eng. 2004;92(1):22–42.[PMC free article] [PubMed] [Google Scholar]
34. Sukhorukov VL, Arnold WM, Zimmermann U. Hypotonically induced changes in the plasma membrane of cultured mammalian cells. J Membr Biol. 1993;132(1):27–40. [PubMed] [Google Scholar]
35. Wang XB, Huang Y, Wang XJ, Becker FF, Gascoyne PRC. Dielectrophoretic manipulation of cells with spiral electrodes. Biophys J. 1997;72(4):1887–1899.[PMC free article] [PubMed] [Google Scholar]
36. Doherty GJ, McMahon HT. Mediation, modulation, and consequences of membrane-cytoskeleton interactions. Annu Rev Biophys. 2008;37:65–95. [PubMed] [Google Scholar]
37. Domnina LV, Ivanova OY, Cherniak BV, Skulachev VP, Vasiliev JM. Effects of the inhibitors of dynamics of cytoskeletal structures on the development of apoptosis induced by the tumor necrosis factor. Biochemistry (Mosc) 2002;67(7):737–746. [PubMed] [Google Scholar]
38. Flusberg DA, Numaguchi Y, Ingber DE. Cooperative control of Akt phosphorylation, bcl-2 expression, and apoptosis by cytoskeletal microflaments and microtubules in capillary endothelial cells. Mol Biol Cell. 2001;12(10):3087–3094.[PMC free article] [PubMed] [Google Scholar]
39. Lai F, Fernald AA, Zhao ND, Le Beau MM. cDNA cloning, expression pattern, genomic structure and chromosomal location of RAB6KIFL, a human kinesin-like gene. Gene. 2000;248(1–2):117–125. [PubMed] [Google Scholar]
40. Sardar HS, Luczak VG, Lopez MM, et al. Mitotic kinesin CENP-E promotes microtubule plus-end elongation. Curr Biol. 2010;20(18):1648–1653.[PMC free article] [PubMed] [Google Scholar]
41. Putkey FR, Cramer T, Morphew MK, et al. Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E. Dev Cell. 2002;3(3):351–365. [PubMed] [Google Scholar]
42. Hübner Y, Hoettges KF, Kass GEN, Ogin SL, Hughes MP. Parallel measurements of drug actions on Erythrocytes by dielectrophoresis, using a three-dimensional electrode design. IEE Proc Nanobiotechnol. 2005;152(4):150–154. [PubMed] [Google Scholar]
43. Yamamoto K, Korenaga R, Kamiya A, Ando J. Fluid shear stress activates Ca2+ influx into human endothelial cells via P2X4 purinoceptors. Circ Res. 2000;87(5):385–391. [PubMed] [Google Scholar]
44. Zhang YJ, Sanchez D, Gorelik J, et al. Basolateral P2X4-like receptors regulate the extracellular ATP-stimulated epithelial Na+ channel activity in renal epithelia. Am J Physiol Renal Physiol. 2007;292(6):F1734–F1740. [PubMed] [Google Scholar]
45. Kobertz WR, Williams C, Miller C. Hanging gondola structure of the T1 domain in a voltage-gated K(+) channel. Biochemistry. 2000;39(34):10347–10352. [PubMed] [Google Scholar]
46. Miller C. An overview of the potassium channel family. Genome Biol. 2000;1(4):Reviews 0004.1–0004.5.[PMC free article] [PubMed] [Google Scholar]
47. Bard AJ, Faulkner LR. Electrochemical Methods: Fundamentals and Applications. 2nd ed. New York, NY: Wiley; 2001. [Google Scholar]
48. Lodish H, Berk A, Zipursky LS, Matsudaira P, Baltimore D, Darnell J. Molecular Cell Biology. 4th ed. New York, NY: W.H.Freeman and Company; 2000. [Google Scholar]
49. Ashley RH. Challenging accepted ion channel biology: p64 and the CLIC family of putative intracellular anion channel proteins. Mol Membr Biol. 2003;20(1):1–11. [PubMed] [Google Scholar]
50. Littler DR, Assaad NN, Harrop SJ, et al. Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4. FEBS J. 2005;272(19):4996–5007. [PubMed] [Google Scholar]
51. Zittoun J, Marquet J, David JC, Maniey D, Zittoun R. A study of the mechanisms of cytotoxicity of Ara-C on three human leukemic cell lines. Cancer Chemother Pharmacol. 1989;24(4):251–255. [PubMed] [Google Scholar]
Articles from International Journal of Nanomedicine are provided here courtesy of Dove Press

. Empire Total War KEYGEN tested

>>> D.o.wn.l.o.a.d Here <<<

Command the seas, control the land, forge a new nation, and conquer the globe. Empire: Total War takes the Total War franchise to the eighteenth century Age .Good day! just passing by your site to read some interesting facts. Thank you for sharing your article, I must say that you did a good job and .Total war attila free download cd key generator king kong games. Empire total war steam key for pc, mac and linux buy now and. Steam key .So I recently brought a new copy of Empire Total War from Amazon.co.uk and and I went to register it and the CD key was already in use.Now I cant play multiplayer and I cant find my CD key. I have looked guys could offer. I am buying Empire soon anyways so it isnt a big deal.I have placed the message in Total war tech issues forums and none answer. Empire Technical Discussion & Support- Duplicate CD key .so i brought the game like 2 maybe 3 years ago i have the case and the disks i have the gold edition but the manual at the back of the case is .Empire: Total War will see the debut of 3D naval combat within the Total War franchise. PC Gamers will be able to intuitively command vast fleets or single ships .If you have previously installed or registered your Empire: Total War keycode to a STEAM account make sure that upon reinstall you.If you are using steam, right click the game in your library and you should be able to select 'view game key', or something to that effect.: buy your CD keys for PC cheapest on the net, and receive them instantly 24/7!.A new detailed North American Campaign Map expands the Empire: Total War experience, with 5 brand new American Indian factions, new units and .Empire: Total War CD KEY (STEAM), empire, total, war, total war, empire total war.COM/empire total war key,empire total war cd key,empire total war code,buy total war key,. Log in or register to reply. Sometimes posts may contain links to .Bought the game used on amazon. Came in great shape, but the cd key is already linked to a steam account. Did I just spend 30 bucks on a .Empire: Total War jetzt als Steam Key kaufen. Herrscht über die Meere, kontrolliert das Land, gestaltet eine neue Nation und erobert die ganze Welt! Empire: .This is a fully legit, Out-Of-The-Box Empire: Total War Steam Key / Serial! Delivery via email within 12 hours of payment. To activate your key, download the .Kinguin offers best deals on Steam CD Keys. Empire: Total War takes the Total War franchise to the eighteenth century Age of Enlightenment .An 'O' is round when printed on the CD key, while a zero has straight i hope that helped i dont have this one all i have is rome and empire.paysafecard:4.79€. Empire: Total War Steam CD Key &middot- Kinguin.net. 4.90 €. paysafecard:5.24€. Empire: Total War &middot- Gameliebe.com. 6.99 €. paysafecard:7.55€.Compare Empire: Total War for Steam prices of digital and online stores. Get Empire: Total War for Steam at the cheapest price. pc cd key best video game .Empire Total War CD Key kaufen - Preisvergleich. Mehr als 20 Shops mit Bewertung: ✓BESTE PREISE ✓Kurze Lieferzeiten. Ein Produkt cd key bei .Prova qui: o qui:.After re-installing to solve a patch problem I realised I no longer have my CD-key. luckily the game accepts any key of the right length for offline .Gdy próbowałem zainstalować Empire Total Wara (na innyjm koncie steam)wyskoczyło ze cd key jest juz używany.Próbowałem się zalogowac .Hallo Community, Heute, zu Ostern habe ich von meiner Mutter das Spiel Empire Total War bekommen. Sie hatte mir erzählt dass sie es erst .App ID, 10605. App Type, Downloadable Content. Name, Empire: Total War - Multiplayer Beta. Last Record Update, 2 years ago (October 7, 2015 – 03:04:28 App ID‎: ‎10605Last Record Update‎: ‎2 years ago (October 7, 2App Type‎: ‎Downloadable Content.Digital copy of the official activation key (CD KEY) game Empire: Total War to activate the system Steam.Empire: Total War keygen Spelunky keygen. oleh Lieconfeirec231. 0 .Comprar Empire Total War Steam CD Key Comparar Precios. Encuentra todos los vendedores de Steam Key para descargar y jugar a Empire Total War al .Download Napoleon: Total War Cd-Key Generator and generate free Product Key! Empire: Total War - The Warpath Campaign, Empire: Total War - Elite Units .Document: empire total war keygen files load by ksd! 15.03.2017 at 16:57.Bonjour à tous voila jai un probleme je me suis fais pierater mon compte steam il y a un moment de cela et javais sur ce compte ma clé cd du .your email. please enter your email. your message. Message goes here. Medieval 2 - Total War. B3D7-FFCA-AB91-4529 S3U6-W8R9-D26N-X34L.EU STEAM CD KEY: Requires an EUROPEAN IP address or you must activate it with a VPN (Virtual Private Network) in order to play Empire Total War The .maar nu ik empire total war wil installeren ,zegt hij natuurlijk dat de ingevoerde product key al Hehe, volgens mij is dat mijn XP serial ook :$.por favor alguien me podria facilitar el serial de empire total war,no lo encuentro por ningun lado.gracias.serial total war. Hola!Tengo un problema, me acaban de dejar el Empire Total War, y al instalarlo mediante el steam, m pide mi contraseña de steam, i la .Empire: Total War for Mac - Factions, Features, Links, Media, Requirements, Steam, MacUser Editors Choice badge MacFormat Choice badge Total War, .480 SHARES Download free Empire: Total War + CD Key FREE! Welcome to our website Game Activation Keys, today we give you FULL GAME with acti.Simpack keys. Thx to Doombuster -g10501 -k6143EE9F1CFDF68B0C2A34DB27A0CD6C ( Empire Total War main) -g10502 .Empire: Total War Multiplayer Beta keys are available! Closed. Mallorn. So I signed up for the new batch of TW Multiplayer beta keys 5 months .For Empire: Total War on the PC, a GameFAQs message board topic the DLC one) and i received a message that i have a duplicate cd-key.if you have an invalid CD-Key, it have to be an illegal CD-Key and the software is illegal. My CD-Key is invalid too, so i have to inform SEGA .Empire total war code generator cd key global game activation. Empire total war gold Empire total war free download with keygen and serial at. Total war attila .Busque por toda la web algun serial o keygen y no encuntro ninguno más que ese que dio darkevans. Alguien que tenga el juego tendría que .Kup GRA PC Empire Total War PL STEAM Cd-Key/Klucz 24h na Allegro – Strona 1 - Najlepsze oferty na największej platformie handlowej.Hello everyone.Ibought Star Wars Empire At War and when I am installing they asked for a CD Key.If anyone has a CD Key plz post it on this .Empire Total War Free Download Empire Total War CD Key Empire Total War Keygen Empire Total War Serial Key. Empire Total War Free Download Empire .Vše o hře Empire + Napoleon Total War pro PC - Steam najdete u nás. Získej informace, komentuj a zjisti aktuální cenu hry.

pro evolution soccer 2011 konami rus fra [repack] ot shepards,Winter Challenge 2008 PC - English TMas,WinRAR 5.0V P+Keygen,WinX DVD Ripper Platinum 6.8.1{h33t}{malestom},兰岛物语-少女的约定,PartyLife MuOnline MAIN (v5.0.4),Mach3VersionR1 83 027 and Crack,U Torrent Full Version Latest by [rAvi],Ashampoo Anti Malware v1.2.1 Incl Keygen and Patch-Lz0,Sony Vegas Movie Studio HD Platinum 11.0.256 Production Suite - Silent,Eurosoft Pc Check 6 00 [CRACKED,TitleTime-Setup,RomaniaKits FifaRo,USDownloader+ updater,BitTorrent9.9,Модный показ 2. Новый сезон,vc-41-i2,USB Disk Security v6 2 0 24 with Key,ibdac40d7pro,Maplesoft Maple v11.0 VISTA-TBE,CCleaner 3.25.1872 With Crack [Tuklu],quick estimator 2005 plus keygen,Zoner Photo Studio Pro v14 0 1 7 + keygen,Norton AntiVirus 2010 v17.0.0.136,Platinum Hide IP v3 0 7 3 Full BiTZEUS,IE8-WindowsXP-x86-CHS,mahjong. pc - kyodai 2006 [keygen],portable all-radio 3.12,Magicka v1.3.3.5 multi4 cracked READ NFO-THETA [Seedoff net],The Tiny Bang Story demo - Help restore Tiny Planet after an asteroid breaks it int,KIS2010-AI,- Powerquest Partiion Magic 8.0 With Full Version Working Serial,hex-editor-neo-ultimate-4.93.03.3402-retail,Atomix Virtual DJ 2008 Professional 5.7 plus All Effects + Key,[SB] Ace Translator Ver. 8.3.0 Build 501,Autocad 2012 x86 (32 bit) Patch 2012 rar,colors-everywhere-screensaver-1.0,Adobe Photoshop CS5 Keygen Patch - TeamZeta,FlashFXP 4.0.0 Build 1510 - Final [H33T],mini-pdf-to-powerpoint-2003-converter-2.0,DVDFab Platinum v8.1.2.9 Qt Beta + Patch,32bit Convert It 10.07.09,Adobe After Effects CS5 - FINAL Version + Keygen

Related Posts[Download] Essential Drivers [Cr.a.ck+][New] Shakira Waka WakaThis Time For Africa [2016 version][Hot] KindlePcConverter331 tested

by mindbracoxprob1971 on 2017-06-14 02:03:44