#7     TISSUE CULTURE TECHNIQUES AND USEFUL PROCEDURES:



1.      Lift cells with trypsin  1.5ml trypsin 10cm or 100-mm dish  15min
37C  +6.5ml complete media  ~8ml


a)      PASSAGE:  Passage ~2.5ml (1/3) of them into new plate for maintaining line


b)      FREEZE LIVE CELLS: Spin 2 tubes-- 1.3ml ea tube-- for freezing live
cell pellet in 500ul bambanker freezing media (Wako) . This is a cool
media that you don't need liquid nitrogen to use! You can freeze cells
in a –80C freezer for years!


c)      COUNT HEMACYTOMETER:  Remainder of cells: 2.5ml  take a 100ul
aliquot to count in hemacytometer 100ul+100ul Trypan blue +100ul PBS
(1:3 dilution)


d)      PCV DETERMINATION:  That is packed cell volume (PCV).
i.      Take 500ul and place in a PCV tube
ii.     spin and get PCV% for each cell line. This PCV tube is a super
cool alternative to always counting cells tediously in a microscope
hemacytometer. MidSci sells it. You can resuse them for the same cell
type by washing with water using a small needle tuberculin syringe
then letting air dry. That unpacks the cell pellet pretty well.