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Media for Hybridoma Fusion, Selection and Maintenance

~craig story

Formulation of media for Myeloma cells

DMEM supplemented with:

  • 10% Fetal Bovine Serum (50 ml) (from media prep freezer)
  • 50 µM 2-Mercaptoethanol (0.5 ml) (make 50 mM stock, filter sterilize)
  • 10 mM HEPES (5 ml) (1:100 of 1M stock Gibco #15630-080)
  • 1 mM Pyruvate (5 ml) (1:100 of 100 mM stock Gibco #11360)
  • 1X MEM Non-Essential Amino Acids (5 ml) (MEM-NEAA) Gibco #11140
  • Penicillin/Streptomycin (5 ml) (100X aliquots in media prep freezer)

Mouse myelomas used

  • NS-1 (ATCC # )
  • SP2/mIL-6 cells (ATCC # CRL-2016)


Media prep for cell fusion (differences from above)

Before mixing up the above medium, remove 50 ml of the DMEM. After removing this medium, add 100 ml FBS from a “hybridoma approved” lot Atlanta Biologicals FBS “Premium Select” lot # S11550 The rest of the formulation is as above Filter the serum or medium prior to fusion to remove particulates from serum that can injure cells.

For resuspending heterokaryons after fusion (HAT selection), add: 1X HAT supplement (purchased from ATCC Cat #69-X) Reconstitute powder with 1.0 ml medium to make a 500X stock 10% Hybridoma Cloning Factor (BioVeris Corporation Cat # 38925 order from Fisher)

Harlow and Lane recommend the following HAT selection sequence: Grow colonies in DMEM 20% FBS + HAT in 96 well tray Keep in HAT when expanding into 24 well tray When cells are dense, expand to 60 mm dish, removing the amethopterin at this stage At this stage it is recommended to add HT (no Amethopterin) to the DMEM until all Amethopterin is diluted out (“several passages”)

HT supplement is ATCC Cat # 71-X

Formulation of media for maintenance hybridoma cells

Same medium as myeloma medium, except it is recommended to use the same lot of bovine serum as cells were selected in for first passages for newly selected cell lines.

Can change to prep room stock FBS once cells are growing well

Medias

supplemented RPMI

RPMI (500ml) supplemented with

  • 10% Inactived Fetal Calf Serum (50 ml)
  • 50 µM 2-beta-Mercaptoethanol (0.5 ml / 50mM filtered solution) (4ul / 14.4M stock)
  • 10 mM HEPES (5 ml / 100X stock)
  • 1 mM Na Pyruvate (5 ml / 100X stock)
  • 1X MEM Non-Essential Amino Acids (5 ml) (MEM-NEAA) Gibco #11140
  • L-glutammine (5ml)
  • Penicillin/Streptomycin (5 ml / 100X aliquots)