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1. Plate 200K cells each well on 6 well plate for O/N cell attachment with 2 mL regular media with 10% FBS.

2. On the next day, remove regular media with 10% FBS, add 2 mL 1XPBS to wash slowly, remove.

3. Add 0.9 mL OptiMEM and 0.1 mL 10x reaction buffer then add the corresponding compounds to the wells.

 Do not do over 12 wells at one time.

4. Treat the cells with the compounds for corresponding hours. Remove the compounds and media, wash with 2 mL 1xPBS slowly, remove.

5. Add 400 ul Trypsin to trypsinize the cells (put in the 37 oC incubator for 10 mins for TNBC cells and 20-30 mins for 22RV1 cells).

6. Add 1 ml regular media to neutralize trypsin. Transfer the whole solution to 1.5 ml Eppendorf tube, centrifuge 300g for 3 mins. 

7. Withdraw the supernatant carefully (leave 50 ul to avoid disregarding the cells).

8. Add 1xPBS and pipette up and down, centrifuge down again.

9. Prepare 4% PFA in 1XPBS, add 0.5 mL 1XPBS first and pipette up and down, then add 0.5 mL 4% PFA in 1XPBS to fix cells. Wait for 10 min at room temperature in dark.

10. Centrifuge down again.

11. Remove PFA. Add 200uL 1% BSA in PBS. Do NOT pipette.

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