Source (Immortomice®) | |||
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Age | Sex | Genotype | Number of mice per preparation |
2–12 weeks, 20 weeks | Female or male | tsSV40Tag heterozygous/homozygous | 2 or 8–16 |
Primary culture (33 or 37 °C) | |||||
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Handling options | Culture media supplements | ||||
Seeding density | Laminin coated surface | Duration until passaging and/or temperature switch | Splitting ratio to passage 1 | Fetal bovine serum (FBS) | Cytosine arabinoside (AraC) |
0.5 × 105–4.5 × 105 cells/cm2 1 × 105 cells/filter limiting dilution cloning (1 cell/0.33 cm2) | Petridishes 35 or 100 mm2 8 well chamberslide 48 well plate 96 well plate | 0–6 days 29/36 days (limited dilution) | 1:2 1:3 1:5 Colony picking | 2 % in the absence of fibroblast growth factor (FGF) 10 % (±FGF) | Days 0–4 |
De-differentiation under permissive conditions (33 °C + IFNγ) | ||||||
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Handling options | Culture media supplements | |||||
Temperature switch | Splitting ratios | Conditioned medium | IFNγ | Fiborblast growth factor (FGF) | Fetal bovine serum | AraC |
With/without splitting of cells | 1:2 1:3 1:4 1:5 1:8 limiting dilution cloning | ±addition of supernatant from HIBCPPs (human choroid plexus papilloma cell line) | 0 U/ml 5 U/ml 10 U/ml 30 U/ml 50 U/ml | ± FGF | 2 % 5 % 10 % | ±AraC days 0–4 |
Re-differentiation under non-permissive conditions (37 or 39 °C) | |
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Handling options | Culture media supplements |
Culture duration | AraC |
7, 10, 14 days | day 0–4 |