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:

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