Preparation of PodoUp3 medium

The PodoUp3 medium for cultivation of PODO/TERT256 and PODO/SVTERT152 cells can either be ordered from Evercyte as ready-to-use medium (Cat#MHT-033-3) or as basal medium (MHT-033-3-B) + Supplements (MHT-033-3-S) or can be prepared by mixing the following components:

take one bottle (500 ml) of MCDB131 (Pan Biotech, Cat# P04-80057)

add 5 ml GlutaMAX-I (200 mM stock, ready-to-use, Gibco, Cat# 35050-038), mix properly

discard 105 ml from MCDB131 / GlutaMAX-I mixture

add 533.6 µl BBE (9 mg/ml stock, ready-to-use, Lonza, Cat# CC-4098)

add 200 µl hEGF stock (20 µg/ml, prepared in cell culture grade water, Sigma-Aldrich, Cat# E9644)

add 200 µl hydrocortisone stock (50 µg/ml, prepared in cell culture grade water, Sigma-Aldrich, Cat# H0396)add 100 ml FBS (ready-to-use, PAN Biotech, Cat# P30-3031)

add 500 µl G418 stock (100 mg/ml, ready-to-use, InvivoGen, Cat# ant-gn-5)

mix properly and store at 4°C for up to 1 month

temper the medium to room temperature (not 37°C) before use

Storage conditions

Store the basal medium at 4°C for up to 6 months and the supplements -20°C for up to 6 months.

Store the ready-to-use medium in aliquots at 4°C for up to 1 month.. Temper the medium to room temperature (not 37°C) before use. Avoid repeated heating to 37°C.

Protocols