An Integrative Procedure for Apoptosis Identification and Measurement
Introduction
Apoptosis is a normal physiological phenomenon put forward by Kerr [1]. It plays an important role in embryonic development, maintenance of tissue homeostasis and pathology, and therefore has become a hotspot of activity in biomedical fields. To date, many methods to detect apoptosis have sprung up, including morphological identification, DNA ladder observation and DNA content and cell cycle analysis. However, these methods differ in treatment and are performed independently, consuming more time. As degraded low-molecular-weight DNA is exuded out of apoptotic cells after fixation with ethanol, DNA dyed with fluorescein will form an apoptosis curve (sub-diploid curve) because its contents are less than that of G1 phase [2]. The quantity of phosphate-citric acid buffer (0.2M, pH7.8) can effectively control the amount of the extracted DNA, and the exuded DNA can be used for DNA ladder detection through gel electrophoresis, while the residual cells can be analyzed with a flow cytometer [3,4]. Here I have uploaded an integrative procedure through which a relatively satisfactory result can be obtained following a single stage of cell culture and transient cell treatment, then detection with different instruments. This shortens experiment time. The protocols are as follows [5-7]:
Materials
Reagents
HEPES (Amresco), RPMI 1640 (HyClone), Penicillin-Streptomycin (Penicillin 10,000units/ml, Streptomycin 10,000µg/ml) (Invitrogen), Fetal bovine serum (FBS) (Sijiqing Co. Hangzhou, China), Trypsin (HyClone), Propidium iodide (Sigma), RNase A (Sina-American Biotechnology Co.) and Proteinase K (Merck).
Any cancer cell line and any apoptosis-inducing drug can be chosen according to the experimenter’s convenience.
Equipment
Incubator, fluorescence microscope, electrophoresis apparatus, flow cytometer and MULTYCYCLE analysis software.
Time Taken
Four days total. (2 overnight incubations, 1 overnight cell fixation and 1 day with 2-3 hours hands-on time)
Procedure
（1） Cell Culture and Induction of Apoptosis
Culture cancer cells in RPMI 1640 supplemented with 10% FBS, 10mM HEPES plus penicillin-streptomycin (100 IU/ml penicillin, 100 µg/ml streptomycin). Induce apoptosis with an apoptosis-inducing drug for 24h. Culture control group cells in medium without drug at 104-6/ml in a flat-bottom plate (Costar).
（2） Cell Collection and fixation
Trypsinize cancer cells（1-2×107）with 1ml 0.25% trypsin at 37℃ for 2~3min until cells detach completely from the flat-bottom. Add 0.5%FBS-RPMI 1640 medium to terminate trypsinization and pipette gently to disrupt cell clumps into single cells. Transfer into an eppendorf tube, spin at 1000 rpm for 5min and discard the supernatant. Wash the cell pellet with 1×PBS at 1000 rpm for 5min×2, resuspend cells into single cell status with a little 1×PBS (20µl), then add 2ml 70% ethanol and place at -20℃ overnight to fix cells.
（3） Centrifuge at 1000 rpm for 10 min; discard the supernatant to remove ethanol thoroughly.
（4） Resuspend cells with 0.5ml 1×PBS, transfer into a micro-eppendorf tube, spin at 1500 rpm for 10 min and discard the supernatant.
（5） Add 40µl 0.2M phosphate-citric acid buffer (pH7.8) to each tube respectively and stand at room temperature for at least 30min with intermitten shaking.
（6） Spin at 1000 rpm for 10 min, transfer the supernatant to new micro-eppendorf tubes and incubate the cell pellets on ice for fluorescence observation and for flow cytometric analysis.
（7） Add 3µl 0.25% NP40 and 3µl RNase A solution（1mg/ml）to the supernatant, vortex the mixture thoroughly and incubate at 37℃ for 30min.
（8） Add again 3µl Proteinase K（1mg/ml）to the mixture，vortex thoroughly and incubate at 37℃ for 30min.
（9） Mix each 16µl reaction solution thoroughly with 2µl 6×DNA loading buffer，and resolve the DNA ladder on a 0.8% 1×TAE agarose gel after electrophoresis at constant voltage 2 V/cm for 2h. Visualize by UV light after standard ethidium bromide staining.
（10） Resuspend the cell pellets in step（6）with 0.5ml 1×PBS, add 10µl Proteinase K (1mg/ml), vortex gently and stand at room temperature for 30mi. Wash with 1ml 1×PBS by spinning at 1000 rpm for 5min×2, resuspend again with 20µl 1×PBS into single cells after discarding the supernatant and then add 300µl DNA staining solution (containing 150µg/ml PI, 20U/ml RNase A). Incubate at room temperature for 30min。
（11） Place a drop of the above single cell suspension on a air-drying glass slide, then place a glass coverslip over the cells to reduce light diffraction and observe cells using a fluorescence microscope equipped with a G.B filter, using the 40×objective. The remaining cell suspension can be used for flow cytometry, and the ratio of apoptotic cells analyzed with MULTYCYCLE software.
