Freezing of A1 cells

in one 175 cm2 flask

Materials:

• APBS (100 ml PBS + 25 ml diwater)
• TE in APBS (36 ml APBS + 4 ml TE)
• 1x 15 ml FALCON tubes
• Pipettes
• 1 cryo tube
• freezing media

Procedure:

1. aspirate the old media from the cells
2. wash the cells 1 times with 5 ml APBS
3. remove the APBS
4. add 5 ml TE
5. wait till the cells are detached
6. stop the TE with HS – media
7. centrifuge at 1000 rpm, 3 min at 4ºC
8. aspirate the supernatant
9. dissolve the pellet into 1 ml freezing media
10. transfer the cells into the cryo tube and label the tube with the cell name, passage number and date
11. store the tube for one day at –80ºC
12. store the vial in liquid N2