Unthawing Cells:

(1) Obtain 2 50 ml tubes; place 10% C RPMI media in to one tube as working stock

(2) before cells are completely thawed, use 2 ml pipette to suck up cells (slowly mix them up and down a couple of times) and then transfer to empty 50 ml tube (let cells slide down the side of tube slowly as you rotate the tube)

(3) Using 2 ml pipette, transfer slowly 2 ml of RPMI media from working stock into tube with cells (do this slowly or cells will burst!). Repeat 4 more times until you have 10 ml total volume.

(4) Using 25 ml pipette transfer more media from working stock to tube with cells for final volume 50 ml

(5) spin 15 min at 1100 RPM (select RPMx1000) (can use big centrifuge outside room)

(6) aspirate with vacuum tube supernatent

(7) transfer 8 ml new media into tube (resuspend pellet by pipetting up and down)

(8) transfer contents of tube to small flask

Changing Media:

Molt-4 cells should be changed every 2-3 days. They grow quicker. maximum [cell] is 2×106 or they will die

(1) transfer cells in flask to tube and spin down

(2) remove supernatent

(3) Wash cells by adding about 50 ml hanks (HBSS) and resuspend pellet (you can add say 5 ml first and then take a 50 ul sample to count cells while spinning cells down)

(4) spin

(5) remove supernatent

(6) resuspend cells in 10 ml of 10% RPMI