Thawing Procedure

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, 2-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 2-mercaptoethanol and 1% 200mM L-Glutamine (100X).

Under normal growth conditions the cells should not be allowed to exceed 1 x 106cells/ml medium.  Cultures are best maintained between 2 x 105 and 8 x 105cells/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.