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Table 1 Culture options tested for the establishment of conditional immortoCPEC lines

From: Modeling immune functions of the mouse blood–cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier

Source (Immortomice®)
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)  
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)
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)  
Handling options Culture media supplements
Culture duration AraC
7, 10, 14 days day 0–4