Test for Mitochondrial Membrane Potential for Cancer cell lines using Mitotracker Red

Materials required:

1) 6-well plates x 7 (2 plates for each cell lines and 1 for control/background)

2) Mitotracker Red (Invitrogen M7513)

3) PBS added with 3% Neonatal Calf Bovine Serum (NCBS) at 37⁰C

4) Accutase

5) Serum free media (0% and 0.5% Serum)

Procedure to prepare stock Mitotracker Red

1) Get the vial (once opened label it with date; only good for a month once opened)

2) Warm it to RTP

3) Add 50µl DMSO

4) Vortex to mix

Procedure for the test:

Day 1 (Monday) (AA, RB, EH)

1) Detach the cells in a 100% confluent flask containing the cancer cell line by adding accutase for

6-10 minutes

2) Add 7ml of complete media to neutralize the action of trypsin

3) Spin the cells down and aspire the supernatant to collect the cell pellets

4) Resuspend the cell pellets in 4ml of complete media

5) In a 50ml tube, add 1ml of the cell suspension to 5.5ml of complete media for CRL-1978 and

CRL-11731. For SK-OV-3, add 1.5ml of cell suspension to 5.5ml of complete media

6) Add 1ml of media to each wells of the 6-well plates

7) Add 1ml of the cell suspension mix to each plate and incubate them at 37⁰C incubator

Day 2 (Tuesday) (AA, RB, EH)

8) Check the 6-well plates to see if the cells are 70-80% confluent. If so, then continue to step 9. If

not, wait until the next day

9) Aspire the old media from each well and add 2ml serum free media (0.5% FBS and L-Glutamine)

and incubate for 6 hours

10) Treat each wells with 20µl of the desired treatment (DMSO, 0.1µM, 0.5µM, 1µM, 5µM, 10µM)

11) Incubate for 24 hours (Until Wednesday)

Day 3 (Wednesday) (AA, WK)

12) Aspire the old media from the wells

13) Add 1ml Accutase to each well and incubate for 6-10 minutes

14) Make 500ml of PBS with 3% NCBS

15) Add 1ml of 3% NCBS with PBS in each well to wash them and collect it in a fax tube

16) Repeat step 11

17) Add mitotracker red (1:100 dilution) to warm (37⁰C) serum free media (0% Serum) to prepare

the staining solution.

Total FACs tubes = 36

Prepare staining solution for 40 tubes

Add 100µl of staining solution in each tube

40 x 100 = 4000µl = 4ml

Add 40µl of the stock Mitotracker red to 4ml of serum free media (0% FBS)

18) Add 100µl of the staining solution in each Fax tubes except the control plate and incubate at

37⁰C for 30-45 minutes

19) Spin the fax tubes down at 500rcf for 5 minutes and aspire the supernatant

20) Add 1ml of 3% NCBS with PBS in each tube to wash the excess stain

21) Repeat step 19

22) Repeat step 20

23) Repeat step 19

24) Resuspend the cell pellet in 300µl of 3% NCBS with PBS and leave it in ice