Love:TCMedia:Cloning

===Formulation of media for expanding hybridomas from single cells===

This media is for expanding single mouse hybridoma cells into larger colonies. Useful for limiting serial dilution or cells picked from microwells.

Note on hybridoma cloning factor (HCF): This media uses hybridoma cloning factor as a replacement for feeder cells. The recommended quantity is 10%. The shelf-life of the cloning factor at 4°C is limited. The recommendation is to thaw the mother stock, aliquot 5 mL portions in 15 mL conical tubes, and refreeze at -20°C. The media below can be prepared in amounts of 50 mL using 5 mL of HCF for 10% v/v. 5% HCF may be sufficient in some cases where you have more than one or two cells retrieved.

Base media: Dulbecco’s Modification of Eagle’s Medium: 1X, with 4.5 gm/L Glucose, with L-Glutamine, with Sodium Pyruvate (Mediatech #45000-304)

Note: Remove appropriate volume of media to account for supplements listed below. Total final volume: ~500 mL

Supplemented with:

• 20% Fetal Bovine Serum (100 ml) (PAA, ###)
• 50 µM 2-Mercaptoethanol (0.5 ml) (make 50 mM stock in PBS or sterile H₂O, filter sterilize)
• 10 mM HEPES (5 ml) (1:100 of 1M stock, Mediatech #15630-080)
• MEM Non-Essential Amino Acids (5 ml) (1:100 of 100x stock, Mediatech #45000-700)
• Penicillin/Streptomycin (5 ml) (100X aliquots in media prep freezer, Mediatech #45000-652)

• Hybridoma cloning factor (10% v/v) (5 mL aliquots in media prep freezer, Bioveris Hybridoma Cloning Factor, Distributed by Fisher, cat #IG-50-0615)

Instructions: Combine 45 mL of DMEM-20%FBS media with 5 mL of HCF in 50 mL conical tube. Use within ~2wks.

(Mediatech products distributed through VWR)