Preparation of ProxUp2 medium

The ProxUp2 medium for cultivation of RPTEC/TERT1 cells contains the herein described components and is prepared as described in the following:

take one bottle (500 ml) of DMEM/F12 (1:1) (PAN-Biotech, Cat# P04-41154)

add 5 ml of HEPES (1M, ready-to-use, Sigma-Aldrich, Cat# H0887)

add 2.5ml of FBS (e.g. PAN Biotech, Cat# P30-3031,)

add 250 µl of hEGF stock (20 µg/ml, prepared in cell culture grade water, Sigma-Aldrich, Cat# E9644

add 250 µl of T3 stock (10 nM, prepared in NaOH/PBS, Sigma-Aldrich, Cat# T6397)

add 250 µl of Ascorbic acid stock (7 mg/ml, prepared in cell culture grade water, Sigma-Aldrich, Cat# A4544)

add 250 µl of Transferrin Holo stock (10 mg/ml, prepared in cell culture grade water, Merck Millipore, Cat# 616424)add 250 µl of Prostaglandine E1 stock (50 µg/ml, prepared in basal medium, Sigma-Aldrich, Cat# P8908)

add 250 µl of Hydrocortisone Stock (50 µg/ml, prepared in cell culture grade water, Sigma-Aldrich, Cat# H0396)

add 250 µl of Sodium-Selenite stock (100 µM, prepared in cell culture grade water, Sigma-Aldrich, Cat# S5261)

add 250 µl of Insulin (10 mg/ml, ready-to-use, Sigma-Aldrich, Cat# I9278)

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

mix properly

store at 4°C for 4 weeks

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