Table 1 |.
Formulations of primary cell culture media
| Component | Stock Concentration | Volume Required for 500 mL (Final Concentration) | |||
|---|---|---|---|---|---|
| Maintenance Medium (MM) | Expansion Medium (EM) | Stem Medium (SM) | Differentiation Medium (DM) | ||
| L-WRN conditioned mediumb | N/A | 250 mL (50 % v/v) | 250 mL (50 % v/v) | 250 mL (50 % v/v) | --- |
| Advanced DMEM/F12 | 1× | 250 mL (50 % v/v) | 250 mL (50 % v/v) | 250 mL (50 % v/v) | 500 mL (100 % v/v) |
| Glutamax | 100× | 5 mL (1×) | 5 mL (1×) | 5 mL (1×) | 5 mL (1×) |
| HEPES | 1 M | 5 mL (10 mM) | 5 mL (10 mM) | 5 mL (10 mM) | 5 mL (10 mM) |
| Primocin | 50 mg mL−1 | 500 μL (50 μg mL−1) | 500 μL (50 μg mL−1) | 500 μL (50 μg mL−1) | 500 μL (50 μg mL−1) |
| N-acetylcysteine | 1 M | 500 μL (1 mM) | 500 μL (1 mM) | 500 μL (1 mM) | 500 μL (1 mM) |
| Epidermal growth factor, murine | 250 μg mL−1 | 100 μL (50 ng mL−1) | 100 μL (50 ng mL−1) | 100 μL (50 ng mL−1) | 100 μL (50 ng mL−1) |
| Nicotinamide | 1 M | --- | 5 mL (10 mM) | --- | --- |
| B27 | 50× | 10 mL (1×) | 10 mL (1×) | --- | --- |
| Gastrin | 1 mg mL−1 | 12.5 μL (10 nM) | 12.5 μL (10 nM) | --- | --- |
| Prostaglandin E2 | 1 mM | --- | 5 μL (10 nM) | --- | --- |
| A 83–01 | 5 mM | 50 μL (500 nM) | --- | 50 μL (500 nM) | 50 μL (500 nM) |
| SB202190 | 30 mM | 50 μL (3 μM) | 50 μL (3 μM) | --- | --- |
| Y-27632 | 10 mM | 500 μL (10 μM) | 500 μL (10 μM)c | --- | --- |
| Heat-inactivated FBS | N/A | 0 mL (10 % v/v) d | 0 mL (10 % v/v) d | 0 mL (10 % v/v) d | 50 mL (10 % v/v) |
a
Stock concentration in parentheses. Refer to Reagent Preparation section for aliquoting instructions.
b
Prepared by culture of the L-WRN cell line (CRL-3276, ATCC, RRID:CVCL_DA06, https://scicrunch.org/resolver/RRID:CVCL_DA06) and harvesting of media containing Wnt3a, R-spondin 3, and noggin. Refer to the protocols published by Stappenbeck and colleagues for details.19,70
c
Y-27632 is always present in MM, though is only included during the initial 48 h of culture in EM.
d
FBS is included in L-WRN conditioned medium at a concentration of 20 %, and no additional FBS is added to MM, EM, or SM. Heat inactivation is conducted in-house by incubating at 56 °C for 25 min.