Culture of mouse hybridomas
Thaw procedure:
Rapidly thaw vial in 37°C water bath, gently agitating vial. Immediately transfer contents of vial to a 75 cm2 flask containing 20 ml medium (Iscove's MDM, 10% FBS, L-glutamine, β-mercaptoethanol). Incubate in 5% CO2/95% air at 37°C.
(A frozen vial of cells will optimally contain approx. 2 - 4 x 106 cells in 1 ml freezing medium.)
Freezing medium:
20% FBS, 10% DMSO in Iscove's MDM.
Growth Medium:
Iscove's MDM (Invitrogen Cat. No. 12440-053), supplemented with 10% Fetal Bovine Serum (Hyclone), 6 x 10-5 M β-mercaptoethanol and 1% 200mM L-Glutamine (100X).
Under normal growth conditions the cells should not be allowed to exceed 1 x 106 cells/ml medium. Cultures are best maintained between 2 x 105 and 8 x 105 cells/ml. Under most bulk antibody production procedures or long term growth conditions, hybridomas should be monitored for populations of cells in the culture that no longer secrete functional antibody.
