1. Pre-coat plates with the coating solution in advance (Matrigel, Geltrex or similar)
*for the room 053 - put autoclaved water to the incubator in one of the plastic boxes
2. Put the growth media and wash media (usually DMEM/F12), which you will use around 30 min in advance to room temperature
3. 10 min before it will be used – warm it in the beads bath
4. Add anti-apoptotic reagent to the media. We usually use pro-survival compound (=PSC, 1000x) or polyamine (1000x) with CET cocktail (4000x) (CET is a mix of Emricasan + trans-ISRIB + Chroman 1)
5.Take out a vial with cells from liquid nitrogen, place it in a box with dry ice and transport it to the cell culture room
6. Thaw the cells quick in a water bath (in the room 053 use the pre-warmed water from the incubator), but leave a small ice crystal in the vial
7. Under the hood, add drop-wise the wash media, approximately 4-5mL
8. Centrifuge for 5 min at 250g
9. Resuspend the cell pellet in warm culturing media with anti-apoptotic
10. Aspirate coating solution or PBS from plates, add cells resuspended in culturing media to 1 well of a 6-well plate
11. Redistribute the cells in the plate and place to the incubator