Hybridoma cells in SFX media

Materials

• 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

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