Induction of Apoptosis
Apoptosis may be induced in response to various cytotoxic stimuli including activation of cell surface receptors such as Fas, TNFR1, TCR, serum or growth factor withdrawl, UV irradiation, various chemicals, glucocorticoid or calcium ionophore treatment, and CTL-targeting of virally or tumor transformed cells. There are numerous methods for induction of apoptosis in vitro and each investigator is encouraged to optimize protocols for their own experimental system. A protocol for induction of apoptosis using camptothecin (a potent inhibitor of topoisomerase I) or staurosporine (a protein kinase inhibitor) is provided in this section.
Preparation of an Apoptotic Jurkat ELISA Standard from Cell Culture
1. Prepare 1.0 mM stock solution of camptothecin (Sigma; Cat. No. C-9911) or staurosporine (Sigma Cat. No. S-4400) in 1.0 mM DMSO.
2 . Add camptothecin (final concentration of 4 -6 µM ) or staurosporine (final concentration of 1-4 µM ) to the cell suspension e.g., 1 x10^6 cells/mL in tissue culture medium.
3. Incubate the cells for 2-18 hr in staurosporine or 4-6 hr in camptothecin at 37°C.
4. After incubation, collect cells and wash with 1X PBS.
5. Prior to use in ELISA, investigators may want to independently confirm that the cells have undergone apoptosis. Please refer to the general scientific literature for methodology.
6. Lyse cells with Cell Lysis Buffer (Cat. No. 559759) for 30 min on ice, vortexing every 5 min. Collect lysate by centrifugation.
7. Determine protein concentration and dilute with Assay Diluent so that the final concentration of the lysate should be at 1 mg/ml. Ideally, fresh lysates should be used for each assay, but the lysates may be aliquoted and stored at -80°C for up to one week.
Reagents Provided
1. Capture Antibody (0.5 ml): Purified Mouse Anti-Human Caspase-3 Antibody (clone 19). For one 96-well plate, add 50 µl Ab to 11.95 ml Coating Buffer. The total volume needed for a 96-well ELISA plate is 9.6 ml, but preparation for a total volume of 12 ml is recommended.
2. Detection Antibody (0.5 ml): Purified Rabbit Anti-Active Caspase-3 Antibody (clone C92-605). For one 96-well plate, add 50 µl Ab to 11.95 ml Assay Diluent. The total volume needed for a 96-well ELISA plate is 9.6 ml, but preparation for a total volume of 12 ml is recommended.
Reagents Not Provided
Note: Do not use sodium azide in these preparations as it will inhibit/inactivate horseradish peroxidase enzyme activity.
1. Enzyme Reagent: HRP Goat Anti-Rabbit Antibody (Cat. No. 554021).
2. Standards: Investigators are encouraged to prepare their own apoptotic lysates.
3. Cell Lysis Buffer (Cat. No. 559759).
4. Coating Buffer: 0.1 M NaHCO3 (pH 9.5). Prepare fresh and use within 7 days of preparation. Store at 2-8°C.
5. Assay Diluent (Cat. No. 555213): Store at 2-8°C.
6. Wash Buffer: Phosphate-Buffered Saline (80.0 g NaCl, 11.6 g Na2HPO4, 2.0 g KH2PO4, 2.0 g KCl, q.s. to 10 L; pH to 7.0) with 0.05% (w/v) Tween-20. Prepare fresh and use within 3 days of preparation. Store at 2-8°C.
7. TMB Substrate Reagent Set (Cat. No. 555214): contains Tetramethylbenzidine (TMB) and hydrogen peroxide.
8. Stop Solution: 1 M H3PO4 or 2 N H2SO4.
9. ELISA/Microtiter plates: BD Falcon™ (Cat. No. 353279) or NUNC Maxisorp (Cat. No. 442404) 96-well plates are suggested.
10. Microplate reader capable of measuring absorbance at 450 nm.
ELISA Assay Protocol
1. Add 100 µl of the mouse anti-human caspase-3 (clone 19) (capture antibody), diluted in Coating Buffer, to each ELISA/microtiter plate well. For one 96-well plate, add 50 µl antibody to 11.95 ml Coating Buffer.
2. Seal plate and incubate overnight at 4° C.
3. Aspirate wells and wash 3 times with ~300 µl/well of Wash Buffer. After last wash, invert plate and blot on absorbent paper to remove any residual buffer.
4. Block plates with ~200 µl/well of Assay Diluent. Incubate at room temperature for 1 hr.
5. Aspirate/wash as in Step 2.
6. Prepare standard and sample dilutions in Assay Diluent.
7. Pipette 100 µl of the standard, sample lysate, and blank (Assay Diluent alone) into appropriate wells. Seal plate and incubate for 2 hr at room temperature.
8. Aspirate/wash as in Step 2, but with 5 total washes.
9. Add 100 µl of rabbit anti-active caspase-3 (clone C92-605) (detection antibody), diluted in Assay Diluent, to each ELISA/microtiter well. For one 96-well plate, add 50 µl antibody to 11.95 ml Assay Diluent.
10. Seal plate and incubate for 1 hr at room temperature.
11. Aspirate/wash as in Step 2, but with 5 total washes.
12. Add 100 µl of HRP Goat Anti-Rabbit antibody (Cat. No. 554021), diluted in Assay Diluent, to each ELISA/microtiter well. For one 96-well plate, add 2.4 µl of antibody to 11.95 ml Assay Diluent.
13. Incubate for 1 hr at room temperature.
14. Aspirate/wash as in Step 2, but with 7 total washes.
15. Add 100 µl of TMB Substrate Solution to each well. Incubate plate, without the plate sealer, for 30 min at room temperature in the dark.
16. Add 50 µl of Stop Solution to each well.
17. Read absorbance at 450 nm within 30 minutes of stopping reaction. If wavelength correction is available, subtract the absorbance at 570 nm from the absorbance at 450 nm.
