TITLE: Conducting Dynamic BH3 Profiling Adapted From Letai Lab
AUTHORS: Dennis Juarez

[DESCRIPTION]
Dynamic BH3 Profiling is used to test cellular proximity to apoptosis after treatment by exposure of in situ mitochondria with the BH3 domain of apoptotic proteins to induce mitochondrial cytochrome C release. Cytochrome C release is determined by flow cytometric measurement of cytochrome C fluorescence in cells and corresponds with the cellular proximity to mitochondrial outer membrane permeabilization, change of which is termed "priming". 

[STEPS]
SECTION: BH3 Peptide Exposure
4. Count your cells and take 2,000,000 cells per treatment condition in a 1.7mL tube use in the assay.
 

SECTION: BH3 Peptide Exposure
6. During the spin, prepare your 2x peptide solutions in MEB2- P25. The peptide stocks are made at 100x and should be diluted 1:50. Prepare enough for every cell line and treatment used in the assay. 

Minimum Total Volume=  (#cell line x #treatment condition + 1)* 25 uL
 

SECTION: BH3 Peptide Exposure
5. Spin down the cells at 500xg for 5 minutes.
SECTION: BH3 Peptide Exposure
8. If you need to stain your cells with cell surface markers, block and stain cells now then spin down before proceeding to digitonin permeabilization. 
 

SECTION: BH3 Peptide Exposure
10. Resuspend the cells in 250uL in the diluted digitonin/MEB2-P25 solution. This is enough for ten 25uL peptide exposures, thus preparing extra.
SECTION: BH3 Peptide Exposure
11. In a V-Bottom plate, place 25uL of 2x peptide at the bottom of the well.
SECTION: BH3 Peptide Exposure
12. Place 25 uL of cells on the wall closest to you.

Gently tap after the addition to each row.
SECTION: BH3 Peptide Exposure
12.1. Mix the 25 uL of cells and 25uL of peptide by gently tapping the sides of the plate. Don't tap to hard or you may lose sample. Proper tapping technique: keep plate on benchtop while tapping. 

Tapping will be necessary in future steps to best mix the solution. Alternatively, a reliable plate vortexer can be used to mix, however inappropriate settings or use may cause solution to spill out of the well.
 

SECTION: BH3 Peptide Exposure
13. Incubate for 1 hr at room temperature. If you have highly sensitive cells, incubate for 30 minutes. Hardier cell lines may require 1.5 hr. Determine this time for your cell system.
SECTION: Ceasing BH3 Exposure and Cytochrome C Staining
14. During incubation, prepare Cytochrome C Stain: 1:40 human cytochrome c (Biolegend) in 10x Cytochrome C Staining buffer, you need 10uL/well, prepare extra.
SECTION: Ceasing BH3 Exposure and Cytochrome C Staining
15. Fix the cells by adding 16.5uL of 8% PFA. This is a final of ~2% PFA.

Gently tap after the addition to each row.
 

SECTION: Ceasing BH3 Exposure and Cytochrome C Staining
16. Incubate at room temp for 20 minutes
SECTION: Ceasing BH3 Exposure and Cytochrome C Staining
17. Neutralize with 16.5uL N2 buffer.

Gently tap after the addition to each row.
SECTION: Ceasing BH3 Exposure and Cytochrome C Staining
18. Wait at least 5 min. 
SECTION: Ceasing BH3 Exposure and Cytochrome C Staining
19. Add 10uL of 1:40 diluted Cytochrome C Staining mix to each well

Gently tap after the addition to each row.
SECTION: Ceasing BH3 Exposure and Cytochrome C Staining
20. Seal the plate with a foil seal or clear seal if kept in the dark.
Incubate for 4 hours at room temperature (in a drawer) or overnight (16hr minimum) at 4 degrees. 
SECTION: Flow Analysis
21. Add ~50uL PBS to increase volume to ~150uL
SECTION: Flow Analysis
22. On the cytometer, open the BH3 Profile Template and BH3 Profiling Instrument Settings
SECTION: Flow Analysis
23. Ensure that the threshold for FSC-H is excluding junk. This assay will have notable amounts of debris.
SECTION: Flow Analysis
24. Run the DMSO control to set cytochrome c high population above the second log
SECTION: Flow Analysis
25. Run the BIM 10uM control to check if cytochrome c low population is below the second log.
SECTION: Flow Analysis
26. Acquire 125uL. Set acquisition settings to acquire 10,000 events.
SECTION: Before Starting
3.1. Testing Digitonin Mediated Cell Permeabilization with Trypan Blue

1. Dilute the digitonin stock to 0.016% in MEB2-P25 and perform a 2-fold serial dilutions five times (0.008%, 0.004%, 0.002%, 0.001%, 0.0005%) to make a panel of 6 different digitonin concentrations (at 2x final) in MEB2-P25 buffer.  

2. Take 400,000 cells you wish to BH3 profile and resuspend in 40 uL of MEB2-P25.

3. Combine 5 uL of cells with 5 uL of the 6 digitonin concentrations.

4. Wait 5 minutes to allow permeabilization

5. Add 10 uL of trypan blue, mix, and transfer to a hemocytometer.

6. Permeabilized cells will appear blue under the microscope.

7. Select the lowest concentration of digitonin that results in 100% permeabilization.
SECTION: Before Starting
3. Determine the minimum concentration of digitonin to permeabilize all your cells. While the Letai BH3 Profiling Guide recommends 0.001% final concentration, this may not work for every cell due to the amount of cholesterol at the cell membrane. Test final digitonin concentrations of 0.008-0.00025%.
SECTION: Before Starting
2. Prepare your peptide panel by diluting your peptides in DMSO to 100x the final concentration desired at exposure. If you do not know the desired final concentration, titrate your peptides to determine a concentration that causes around 20% cytochrome c release.
 
 
        Peptide  Exposure Concentration  (uM)    BIM  0.001-100    BID  0.01-100    PUMA  0.1-100    BAD  0.1-100    NOXA  1-100    HRKy  1-100    MS1  0.1-10    FS1  0.1-10    ABT199  0.1-1    WEHI-539  0.1-1    A-1331852  0.1-1    A-1155463  0.1-1    S63845  1-10  

SECTION: Before Starting
1. Allow the MEB2-P25 to equilibrate to room temperature (RT) if stored at 4 degree. Equilibration may take 1 to 2 hours. Preferably, you can pull the bottle out the night before to allow RT equilibration.
 

SECTION: BH3 Peptide Exposure
9. During this step, dilute the digitonin to 2x of the final concentration determined in step 3.1 in MEB2-P25 buffer.
SECTION: BH3 Peptide Exposure
7. Aspirate media and resuspend with 1mL PBS and spin down in cold centrifuge to wash the cells.