Measuring Multiple Apoptosis Parameters with the Agilent 2100 Bioanalyzer Application
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Measuring multiple apoptosis parameters with the Agilent 2100 bioanalyzer A simplified and fast protocol for the analysis of DNA fragmentation during apoptosis Application Marc Valer Abstract This Application Note describes a simplified and fast protocol for apop- tosis DNA fragmentation analysis with the Agilent 2100 bioanalyzer using the DNA LabChip® kit. The combination of Annexin V, active- caspase-3 and DNA laddering analysis on the same platform together with the Cell Fluorescence LabChip kit permits quick confirmation and quantitation of apoptosis in cell populations. A fast and simple protocol was applied for the extraction of fragmented, chromosomal DNA. The increased sensitivity and sizing accuracy of the DNA LabChip kit com- pared to conventional agarose gel electrophoresis also means that a significant lower amount of apoptotic cells is required for the analysis. Introduction research scientists in a wide vari- ety of fields. Apoptosis is charac- The Agilent 2100 bioanalyzer was terized by a distinct set of mor- introduced by Agilent Technolo- phological events involving plas- gies as the first commerially avail- ma membrane blebbing and asym- able lab-on-a-chip analysis system metry loss, reduction of cell vol- for the life science laboratory ume, loss of mitochondrial mem- using LabChip® products, devel- brane potential, nuclear condensa- oped in collaboration with Caliper tion, fragmentation of DNA at Technologies Corp. Chip-based nucleosomal intervals and other approaches for a variety of separa- cytoplasmatic changes. Ultimately tion-based techniques have been fragmentation of the cell into introduced, addressing DNA, RNA membrane-enclosed “apoptotic and protein separations.1, 2, 3. bodies” occurs. Differential Recently the Agilent 2100 bioana- expression patterns or alternate lyzer has been extended to enable mechanisms in the apoptosis path- dual color simple flow cytometric ways among tissues and cell lines assays4. Apoptosis assays have makes it necessary to have two or been demonstrated for quantita- even three parallel experiments to tive measurements of whole cells5. confirm, quantitate and compare The combination of cytometric kinetic events on apoptosis. and gel-like separations on the same instrument platform makes The appearance of DNA laddering it an ideal tool for multi-paramet- is unambiguously connected to ric apoptosis quantitation and con- apoptosis. Targeting of phos- firmation. phatidyl serine (PS) in the outer leaflet of cell membrane with fluo- Apoptosis rescently labeled annexin V and Apoptosis, or programmed cell antibody labeling of active-cas- death, is the outcome of a meta- pase-3 in cells has been previously bolic cascade that results in cells described in conjunction with the dying in a controlled manner. It is Agilent 2100 bioanalyzer as a used by nature during develop- quantitative measurement for 5 ment6, homeostasis, aging and in apoptotic cell samples . defense mechanisms. Apoptosis is a key factor in cancer and also This Application Note describes suspected to be a characteristic the combined measurement of mechanism for degenerative disor- annexin V and the extracted DNA ders such as Alzheimer7 or Parkin- of apoptotic cells on one platform son's diseases. The process is uni- – the Agilent 2100 bioanalyzer. A versal and has been described in fast protocol for DNA preparation eukaryotic cells as well as in indi- and recommendations for data vidual bacteria8, protozoa9 and evaluation are discussed. amoeba10. It is a target for 2 Experimental *As an alternative to the PCR purification kit, the following pro- Apoptosis was induced in Jurkat tocol has been successfully tested: cells by incubation in a 5 µM solu- tion of the topoisomerase • Add 2 µL 5M NaCl to the lysate inhibitor Camptothecin (Sigma, # and vortex 5 seconds. C9911) in medium. Cells were har- • Add 200 µL ice-cold absolute vested and counted at the indicat- ethanol and vortex 5 seconds. ed time intervals. Cell lysis and • Incubate on ice for 10 minutes DNA purification was performed • Centrifuge at 13000 g for 5 min- as described in the fast protocol utes. Discard supernatant. below. Eluted DNA was directly • Dry residual ethanol in speed- loaded into the chip wells, pre- vac or desiccator. pared according to the LabChip • Resuspend precipitate in 30 µL Reagent Kit Guide. of pre-warmed (65 °C) distilled water or 10 mM Tris/10 mM Fast protocol for DNA fragment EDTA. purification (based on original • Run in Agilent 2100 bioanalyzer protocol in reference 11) with the DNA 12000 LabChip • Pellet 1-2 × 106 cells by cen- kit, selecting the DNA 12000 trifugation (2 min × 400 g). laddering assay. Remove medium. • Add 100 µL 4 °C lysis solution UV measurements (0. 2% Triton X, 10 mM Tris, 10 UV spectroscopy readings were mM EDTA) and gently resus- performed to determine total DNA pend. concentration. The Agilent 8453 • Incubate 5 min at 4 °C. UV-visible spectrophotometer was • Centrifuge for 5 min at 13000 g. used in conjunction with a 0.2-cm • Transfer supernatant to new light path ultra-micro cell (10 µl vial and discard the pellet. volume). The whole spectrum • Purify DNA using a low volume (190–1100 nm) was acquired and elution method. Here a PCR evaluation was automatically done purification kit* was used by the Warburg-Christian (QIAGEN MinElute PCR Purifi- method12 with internal wave- cation Kit. Cat.28004). length correction at 320/10 nm. • Run in Agilent 2100 bioanalyzer with the DNA 12000 LabChip Annexin V apoptosis measurements Kit, selecting the DNA 12000 A fast annexin protocol as Laddering assay from the bio- described in reference 13 was sizing software. used. 3 Results and Discussion A. Agilent 2100 bioanalyzer cell assay Besides DNA laddering, cytomet- ric assays like annexin V or cas- pase analysis are commonly used for apoptosis detection. Figure 1A shows the analysis of annexin V in Jurkat cells on the Agilent 2100 bioanalyzer after 6 hours treat- ment with 5 µM campthotecin. After switching the instrument to electrophoresis mode, extracted DNA from treated Jurkat cells was analyzed using the DNA 12000 LabChip kit. The characteristic DNA laddering further confirmed apoptosis in the sample (figure 1B). Sizing information, provided by the 2100 bioanalyzer software, shows nucleosomal fragmentation of DNA by evenly distributed peaks in the electropherogram (figure 2). Apoptosis is confirmed B. Agilent 2100 bioanalyzer DNA assay in samples by fragmentation of the chromosomal DNA in 180–200bp Fluorescence intervals.14 150 140 Typical DNA extraction with the described fast protocol leads to 130 10 µL of 2-30ng DNA/µL (figure 3). 120 The DNA 12000 concentration range specification is 0.5–50 110 ng/µL, so no further treatment is 100 required. Band concentration was 90 calculated to be approximately 1 ng/µL (1 µL loaded on the chip). 80 70 25 30 35 40 45 50 55 60 65 70 75 Time [s] Figure 1 A. Histogram view of the two-color flow cytometric analysis showing annexin V-biotin, strepta- vidin-Cy5® staining of phosphatidyl serine in untreated (upper panel) and apoptosis induced Jurkat cells (lower panel). Left panels show reference staining with calcein for life cells, as annexin also stains cells with a damaged membrane, e.g. necrotic and dead cells. B. Apoptosis confirmation by DNA laddering assay, electropherogram and gel like image are shown. 4 Fluorescence Peak Mig.Time(secs) Size(bp) Marker 1 35.4 50 Lower 100 2 41.4 169 169 3 50.7 353 184 4 58.2 535 182 90 5 63.6 710 175 6 67.4 906 196 80 7 69.9 1106 200 8 72.9 1667 561 9 83.0 10380 Upper 70 Figure 2 60 Electrophrogram of U937 cells lyophilized positive control (Roche diagnostics). Automat- 50 ed size calibration allows a quick verification of DNA fragmentation sizes. DNA peaks at 710 50* 10380* 906 353 1106 1667 169 40 535 180–200bp intervals were well according to the expected values for nucleosomal 25 30 35 40 45 50 55 60 65 70 75 80 85 90 fragmentation. Time[s] Absorbance (AU) 0.03 Absorbance (AU) 0.02 0.01 0.5 0 220 240 260 280 300 320 0.4 Wavelength (nm) 0.3 0.2 0.1 0 220 240 260 280 300 320 Wavelength [nm] Figure 3 Accurate UV quantitation of DNA was Necrotic control Sample 4 Unit achieved with 5 µL of sample (diluted to J 20H 10 % ETOH J 48H 5 µm CAM 10 µL with water). The high sensitivity of the Agilent 8453 spectrophotometer in combina- 260 2.7E-02 0.54 AU tion with an ultra micro-cell provides high quality spectra even at very low absorbance 280 1.5E-02 0.27 AU levels (spectrum magnification). The UV spec- 320 1.1E-04 0.00 AU tra with higher absorbance (red) corresponds Warburg-Christian 2.3 24.0 ng/µL to the apoptic sample 4 (figure 4). The spectra Protein 4.7 17.0 ng/µL with lower absorbance (blue) corresponds to the necrotic control sample. 5 Even at low concentrations, apop- totic samples are clearly distin- guishable from the negative con- trol and necrotic samples (figure 4, samples 1 to 7). The positive samples in figure 4 turned out to be 35 % apoptotic as confirmed by the annexin V assay. A clear dis- crimination of samples that con- tain a lower number of apoptotic cells is achieved by overlaying negative controls and samples in the electropherogram view Figure 4 (figure 5). Gel-like image showing positive and negative controls together with 4 apoptotic samples. Negative and necrotic samples display an overall smaller DNA signal and no trace of laddering. If alternative DNA purification methods are used, special atten- tion must be paid as the injection of large amounts of genomic DNA Fluorescence (like whole cell lysates) may lead 40 to clogging of the chip.