Hybridoma cells in SFX media: Difference between revisions

Latest revision as of 14:35, 8 June 2015

5 ml, 10 ml, 20 ml pipettes
Serum free media (400 mls SF media + 1% antibiotics; 4 mls P/S and 4 mls Glutamine)
2 blue cap flasks
1 conical tube

fill 10 mls SFX media per blue cap flask
put flask for > = 1 hour in incubator 10% CO2 @ 37 °C
use 10 ml pipette to take up the media and blow off the cells
check the flask in microscope to make sure that the cells have detached
use 10 ml pipette to remove the cells from the flask into the conical tube
spin at 1000rpm for 3 minutes
aspirate off the media
add 2 mls of SFX media
resuspend the cells
add the 2 mls of the suspension to 1flask
ut in 10% CO2 incubator
let cells grow until they die ( 1-2 weeks; spin cells down( 1000 rpm , 3 min. 4°C)
collect supernatent( contains Antibody) and add @1:50 concentratin 5 x sodium azide/ 500 mM Hepes and store at 4°C

@@ Line 1: / Line 1: @@
-==Materials:==
+== Material ==
-<li>5 ml, 10 ml, 20 ml pipettes
+* 5 ml, 10 ml, 20 ml pipettes
-<li>Serum free media( 400 mls SF media  + 1%   antibiotics
+* Serum free media (400 mls SF media  + 1%   antibiotics; 4 mls P/S and 4 mls Glutamine)
-mls P/S and 4 mls Glutamine
+* 2 blue cap flasks
-<li>2 blue cap flasks
+* 1 conical tube
-<li>1 conical tube
+== Procedure ==
+# fill 10 mls SFX media per blue cap flask
+# put flask  for > = 1 hour in incubator 10% CO2 @ 37 °C
+# use 10 ml pipette to take up the media and blow off the cells
+# check the flask in microscope to make sure that the cells have detached
+# use 10 ml pipette to remove the cells from the flask into the conical tube
+# spin at 1000rpm for 3 minutes
+# aspirate off the media
+# add 2  mls  of SFX media
+# resuspend the cells
+# add the 2 mls of the suspension to 1flask
+# ut in 10% CO2 incubator
+# let cells grow until they die ( 1-2 weeks; spin cells down( 1000 rpm , 3 min. 4°C)
+# collect supernatent( contains Antibody)  and add  @1:50 concentratin 5 x sodium azide/ 500 mM Hepes and store at 4°C