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