Difference between revisions of "Hybridoma cells in SFX media"
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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
(Created page with "==Materials:== <li>5 ml, 10 ml, 20 ml pipettes <li>Serum free media( 400 mls SF media + 1% antibiotics 4 mls P/S and 4 mls Glutamine <li>2 blue cap...") |
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Line 5: | Line 5: | ||
<li>2 blue cap flasks | <li>2 blue cap flasks | ||
<li>1 conical tube | <li>1 conical tube | ||
+ | |||
+ | ==Execution:== | ||
+ | # 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 |
Revision as of 11:23, 4 August 2014
Materials:
Execution:
- 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