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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