Apoptosis induction

Measurement of cytochrome C release from mitochondria upon induction of intrinsic apoptosis with raptinal

(adapted from Abcam ab109719 protocol booklet)

Apoptosis induction

1. Seed 1x10⁶ HeLa cells (ATCC CCL-2) per well into four wells of a six-well tissue culture-treated plate (Greiner 657165) with 2 mL Dulbecco’s Modified Eagle Medium (DMEM; ThermoFisher 12800082) supplemented with 10% fetal bovine serum (VWR 89510–186) and 1X penicillin-streptomycin (ThermoFisher 15140122). Incubate cells overnight and perform subsequent treatments at 37 °C with 5% CO₂ .
2. Pre-treat two wells each with 20 μM caspase inhibitor zVAD-FMK (zVAD; Promega G7231) or equal volume of vehicle (DMSO) for one hour.
3. Treat two wells each with 10 μM raptinal (MilliporeSigma SML1745) or equal volume of DMSO for four hours, for a total of four unique conditions: DMSO only, DMSO and 10 μM raptinal, 20 μM zVAD and DMSO, 20 μM zVAD and 10 μM raptinal. (Note: do not exceed 0.1% DMSO in any well to avoid toxicity.)

Cell fractionation

1. During zVAD and raptinal treatment, equilibrate 2X Buffer A from Cell Fractionation Kit - Standard (Abcam ab109719) to room temperature (RT).
2. Carefully remove media from each well into 2 mL microcentrifuge tubes and pellet detached cells at 300 x g for five minutes at RT.
3. Wash attached cells in each well with 4 °C Dulbecco's phosphate-buffered saline (ThermoFisher 14190-250), then add 200 μL 0.25% Trypsin-EDTA (ThermoFisher 25300120) to each well. Incubate at 37 °C for five minutes.
4. Discard cell pellet supernatants from 2 mL microcentrifuge tubes, wash trypsinized cells off plate with 1.8 mL DMEM, and add trypsinized cells to each pellet from the corresponding well. Pellet cells again at 300 x g for five minutes at RT.
5. During centrifugation, add 2 mL 2X Buffer A to 2 mL nuclease-free water to make 1X Buffer A.
6. Discard cell pellet supernatants and re-suspend each pellet in 150 μL 1X Buffer A.
7. Immediately prior to use, dilute Detergent I 1000-fold in 1X Buffer A to make Buffer B.
8. Transfer 135 μL of each cell suspension to a new 1.5 mL microcentrifuge tube, add an equal volume of Buffer B, and mix by pipetting. Incubate samples for seven minutes on a rotator at RT.
9. Centrifuge samples at 5,000 x g for one minute at 4°C. Carefully remove all supernatants and transfer them to new 1.5 mL microcentrifuge tubes. Place pellets on ice. Re-centrifuge the supernatant fractions at 10,000 x g for one minute at 4 °C.
10. Transfer the supernatants containing cytosolic proteins into a new set of tubes.
11. Re-suspend and combine the sequential cytoplasm-depleted pellets in 150 μL 1X Buffer A.
12. Immediately prior to use, dilute Detergent II 25-fold in 1X Buffer A to make Buffer C.
13. Transfer 135 μL each cytoplasm-depleted suspension to new tubes, add an equal volume of Buffer C, and mix by pipetting. Incubate samples for 10 minutes on a rotator at RT. 
14. Centrifuge samples at 5,000 x g for one minute at 4 °C. Carefully remove supernatants and transfer them to new tubes. Re-centrifuge the supernatant fractions at 10,000 x g for one minute.
15. Transfer the resulting supernatants containing mitochondrial proteins into a new set of tubes. Nuclear pellets may be discarded or suspended in 1X Buffer A for downstream analysis.

Protein quantification, SDS-PAGE, and Western blotting

1. Quantify each cytoplasmic and mitochondrial fraction using a standard colorimetric protein assay such as Bradford, BCA, or Lowry.
2. Add 1/4th volume of 5X SDS-PAGE sample buffer to 10 μg of each fraction, boil samples for five minutes at 100 °C, then place on ice for five minutes.
3. Run samples alongside a prestained protein ladder (ThermoFisher 26616) on a SDS-PAGE gel until the dye front reaches the bottom of the gel. 
4. Transfer gel to nitrocelluose membrane and cut membrane into sections surrounding proteins of interest (such as cytochrome C) as well as appropriate cytoplasmic and mitochondrial loading controls (such as α-tubulin and VDAC1, respectively).
5. Analyze protein expression by western blot using primary antibodies raised against the protein(s) of interest. Suggested antibodies for measurement of cytochrome C release (RRID, dilution): rabbit monoclonal anti-cytochrome c (CYTC) antibody (AB_2637071, 1:500), mouse monoclonal anti-alpha tubulin (AB_2241126, 1:500), and rabbit polyclonal anti-VDAC1/Porin (AB_2214787; 1:1000).