Difference between revisions of "Hybridoma cells in SFX media"
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(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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| − | == | + | == Material == |
| − | + | * 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 | |
| − | + | ||
| + | == 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 | ||
Latest revision as of 14:35, 8 June 2015
Material
- 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
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