These are the materials needed:
100 cm culture dishes
T75 culture flasks
500 cm culture dishes – 166508 - Standard Height, Nunclon Delta Treated Square BioAssay Dish
PA-1 culture media
- 450 ml MEM + GLUTAMAX (GIBCO X10, 42360032 – 42360099x10)
- 50 ml 10% SBF (PAA, A15/043). Heat inactivated (20min a 56ºC)
- 5 ml of 1% Penicilin/Streptomicin 100 ml (100x). (GIBCO, 15140122)
- 5 ml of 1% MEM-NEAA (GIBCO, 11140-035)
- Only if MEM doesn't include Glutamine: 1% GLUTAMAX-1 SUPPLEMENT (GIBCO, 35050-038) o L-GLUTAMINA 200mM (100x) 20ML (GIBCO, 25030-032) (in case the media doesn’t have L-Glutamine)
PA-1 Differentiation culture media
- 450 ml 450 ml MEM + GLUTAMAX (GIBCO X10, 42360032 – 42360099x10)
- 50 ml KnockOut Serum replacement medium (10%, KOSR, Invitrogen, 10828028)
- 1µM of retinoic acid (RA, trans-only R2625-100MG, Sigma)
- 5 ml of 1% Penicilin/Streptomicin 100 ml (100x). (GIBCO, 15140122)
- 5 ml of 1% MEM-NEAA (GIBCO, 11140-035)
- Only if MEM doesn't include Glutamine: 1% GLUTAMAX-1 SUPPLEMENT (GIBCO, 35050-038) o L-GLUTAMINA 200mM (100x) 20ML (GIBCO, 25030-032) (in case the media doesn’t have L-Glutamine)
N2102Ep Clone 2A6
A non seminomatous germ cell carcinoma derived cell line. This cell line was established from a single cell clone from a subline of cell line 2102Ep passaged through a nude mouse. This cell line is tumourigenic in nude mice.
Culture medium
DMEM (High Glucose) (No sodium pyruvate) + 2mM Glutamine + 10% Foetal Bovine Serum (FBS):
500 ml media
50 ml FBS
5 ml of Glutamine 100 mM (100X)
5 ml of pen/strep 10 000 mM (100X) – Final conc 100 U/ml
NTERA-2 cl. D1
The parental NTERA-2 lines was established in 1980 from a nude mouse xenograft of the Tera-2 cell line (see ATCC HTB-106).
The NTERA-2 cl.D1 cell line is a pluripotent human testicular embryonal carcinoma cell line derived by cloning the NTERA-2 cell line.
Culture medium
DMEM (High Glucose) + 4mM Glutamine + 10% Foetal Bovine Serum (FBS).
The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10% and penicilin/streptavidin (100 U/ml)
500 ml media
50 ml FBS
5 ml of pen/strep 10 000 mM (100X) - Final conc 100 U/ml
- DPBS 1X
- TRIPSINA-EDTA (1X), 0.05%, 500ML (GIBCO, 25300-062)
- TrypLE™ Express Enzyme (1X), no phenol red (GIBCO, 12604021)
- Matrigel (Basement Membrane Matrix 10 ml * LDEV-Free, BD 354234)
Descomplemented FBS with DMSO 10%
X-tremeGENE™ 9 DNA Transfection Reagent - https://www.sigmaaldrich.com/catalog/product/roche/xtg9ro?lang=en®ion=US
Optimem (GIBCO, 31985-047)
Protocols:
Thawing Frozen Cells
Cell Passage
Freezing Cells
Cell transfection
PA-1 differentiation