KT 01/06/06
Plating HT22 Cells
1) Warm T/E and HT22 media in water bath
2) Turn flask on side & suck out all media
3) Add 5mL of T/E (in histology freezer)
4) Incubate for 3-5minutes until cells are floating
5) Meanwhile add 400uL of trypan blue to eppitube
6) Add 5mL of media to flask – mix gently
7) Suck off cells with 10mL pipette & place in 50mL conical tube
8) Centrifuge conical tube for 10min @ 3000RPM in Levitt lab centrifuge
9) Suck off media via suction w/ glass pipette
10) Add 5mL HT22 media into conical tube
11) With glass pipette & bulb, pipette up and down to break up cell suspension
12) Set up hemacytometer
13) Pipette 100uL of cells into the 400uL of trypan blue
14) Mix cells gently in trypan blue – place 21uL in hemacytometer
15) Count all 8 quadrants to give the cell count – calculations as follows
(Cell count divided by # of quadrants) x 5 (1:5 dilution) x 10,000 (hemacytometer
correction) divided by (concentration/mL that you want) = X
[(Amount to be plated) mL divided by X from above] = # of cells from conical tube
to use
Example:
(88/8) x 5 x 10,000 divided by 30,000cells/mL = 18.33
need 15mL divided by 18.33 = 0.818mL of cells
Therefore you add 0.818mL of cells to 14.18mL of media to = 15mL