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This protocol has not been updated this year! Last update was on April 01, 2025.
TC media recipes
N3 media
Used for neuronal differentiation during direct conversion.
Total volume: 500 mL
Component |
% |
Volume |
|---|---|---|
Glutamax (1%) |
1% |
5 mL |
N2 (100x stock) |
100x stock |
5 mL |
B27 (50x stock) |
50x stock |
10 mL |
Pen/Strep (1%) (optional) |
1% |
5 mL |
FBS (optional) |
2% |
10 mL |
DMEM F12 |
500 mL |
Add the following small molecules to small volumes immediatly before media addition, to a final concentration of 10 ng/mL:
BDNF, from 1,000x stock (10,000 ng/mL)
CNTF, from 1,000x stock (10,000 ng/mL)
GDNF, from 1,000x stock (10,000 ng/mL)
FGF, from 10,000x stock (100,000 ng/mL)
RepSox, if RR, from 1000x stock (7.5mM)
Note
200mM glutamine is the same as Glutamax
We do not usually use FBS or Pen/Strep
Wrap N3 in foil after making
MEF media
Used for culturing MEFs and other fibroblasts.
Total volume: 500 mL
Component |
% |
Volume |
|---|---|---|
FBS |
10% |
50 mL |
Pen/Strep (optional) |
1% |
5 mL |
DMEM |
add to 500mL |
Breast cancer 1 media
Use for culturing the LM2, BT-20, and MDA-MB-468 cell lines.
Component |
Concentration |
Volume / 50 mL |
|---|---|---|
DMEM + 10% FBS |
Base |
Use MEF media |
Anti-Anti |
1% |
500 uL |
Breast cancer 2 media
Use for culturing the HS 578T and BT-549 cell lines.
Component |
Concentration |
Volume / 50 mL |
|---|---|---|
DMEM + 10% FBS |
Base |
Use MEF media |
PSG |
1% |
500 uL |
Insulin |
10 ug/mL |
125 uL of 4 mg/mL stock |
Breast cancer 3 media
Use for culturing the MDA-MD-231 cell line.
Component |
Concentration |
Volume / 50 mL |
|---|---|---|
DMEM + 10% FBS |
Base |
Use MEF media |
PSG |
1% |
500 uL |
Breast cancer 4 media
Use for culturing the SUM159 cell line.
Component |
Concentration |
Volume / 50 mL |
|---|---|---|
F12 |
Base |
47 mL |
FBS |
5% |
2.5 mL |
Anti-anti |
1% |
500 uL |
Insulin |
5 ug/mL |
62.5 uL of 4 mg/mL stock |
Hydrocortisone |
1 ug/mL |
|
EGF |
20 ng/mL |
1 uL of 1 mg/mL stock |
Epithelial cell 1 media
Use for culturing the NCI-H1299 human lung cancer cell line.
Component |
Concentration |
|---|---|
RPMI + 10% FBS |
Base |
Epithelial cell 2 media
Use for culturing the SAOS-2 human osteosarcoma cell line.
Component |
Concentration |
|---|---|
McCoy’s + 15% FBS |
Base |
Glia media
Used for culturing glia cells
Total volume: 500 mL
Component |
% |
Volume |
|---|---|---|
Horse serum |
10% |
50 mL |
Glucose |
20% |
100 mL |
MEM |
70% |
350 mL |
Sorting/Collection media
Used for cell sorting. Use DMEM/F12 for flow sorting and DMEM/F12 + 10% FBS for collection to help make cells happier. It is possible to use whatever though because you will have to spin-down and resuspend in the correct media (i.e. N3) anyways. You will want to include Pen/Strep as it will greatly reduce the amount of contamination.
Total volume: 50 mL
Component |
% |
Volume |
|---|---|---|
FBS (for collection only) |
10% |
5 mL |
DMEM/F12 |
89% |
44.5 mL |
100X Pen/Strep |
1% |
500 µL |
Motor neuron dissociation media
Used for dissociating iMNs or primary motor neurons (embMN) harvested from spinal cords for plating/sorting. Add 50 µL/96-well, let sit in 37°C for ~15 min. Lightly tap plae to see if cells are dissociating.
Total volume: 6 mL (enough for 1x96-well)
Component |
Volume |
|---|---|
Papain |
1 vial (>= 100 U/vial) |
DNAse |
1 vial (>= 1,000 U/vial) |
DMEM/F12 |
6 mL |
Freezing media
Component |
Volume (1 mL) |
Final Concentration |
|---|---|---|
FBS (or DMEM/10% FBS) |
900 µL |
90% |
DMSO |
100 µL |
10% |
It is easy to keep a 4°C stock of (80% FBS/20% DMSO) then use 500 µL 80%/20% FBS/DMSO + 500 µL DMEM/FBS cell solution
HEPES-buffered DMEM
For use during lentivirus or retrovirus production in HEK293T cells. We use HEPES Sigma-Aldrich (H3375).
1M HEPES stock solution (filter sterilize with 0.22 µm filter after pH-ing)
Component |
Concentration |
Amount/50 mL |
|---|---|---|
HEPES |
1M |
13.82 g |
DI H2O |
main solvent |
50 mL |
NaOH |
to pH 7.0 |
Note
When NBW did this on 7/10/23, I did 41.46 g HEPES + 150 mL water. The HEPES takes up a lot of volume so I would dissolve in 100 mL ELGA-filtered water, then check volume and increase to 150 mL.
At the beginning, pH = 5.45 and I had to add 8.9 mL 4N NaOH to get to pH = 7.00.
HEPES-buffered DMEM
Component |
Concentration |
Amount/50 mL |
Bottle |
|---|---|---|---|
DMEM + 10% FBS |
main Component |
48.75 mL |
550 mL |
Sterile 1M HEPES |
25 mM |
1.25 mL |
14.10 mL |
iPSC Reprogramming Media
Used for reprogramming fibroblasts to iPSCs.
Last updated: 2025-04-01
From: Hoetker, Michael S., et al. “H3K36 methylation maintains cell identity by regulating opposing lineage programmes.” Nature cell biology 25.8 (2023): 1121-1134.
Total Volume: 50 mL
Component |
% |
Volume |
|---|---|---|
KO-DMEM |
1x stock |
41.4 mL |
de-activated FBS |
15% |
7.5 mL |
Glutamax |
1% |
500 uL |
MEM/NEAA |
1% |
500 uL |
Leukemia inhibitory factor (LIF) |
1000x stock |
50 uL |
β-mercaptoethanol |
1000x stock |
50 uL |
Note
β-mercaptoethanol is not stable in dilute solution, so add fresh to an aliquot of media right before each media change. As of 2025-03-24, LIF is also aliquotted fresh from -20°C (do not refreeze).
50 mM β-mercaptoethanol stock solution
Total volume: 1.2 mL (enough for ~20 aliquots of 60 uL)
Component |
Concentration |
Volume |
|---|---|---|
Starting stock |
14.3 M |
4.2 uL |
PBS |
1x |
1195.8 uL |