Biology Reference
In-Depth Information
NaOH, if necessary. Make up the volume to 1.0 L, sterilize by
fi ltering or autoclaving (20 min, 15 psi, liquid cycle), and store
at room temperature.
10. 100× Collagenase-dispase solution: dissolve 100 mg collage-
nase-dispase powder in 1 mL PBS. Filter-sterilize and store in
aliquots at −20 °C.
11. 2×
N
-2-Hydroxyethylpiperazine-
N
-2-ethanesulfonic acid
(HEPES)-buffered saline (HeBS): dissolve 8.2 g NaCl, 5.95 g
HEPES, and 105 mg Na
2
HPO
4
in 400 mL double-distilled
water and adjust pH to exactly 7.05. The exact pH is extremely
important for effi cient transfection (the optimal pH range is
7.05-7.12). Add water to 500 mL, fi lter-sterilize, and store in
aliquots at −20 °C. Avoid repeated freezing and thawing. Test
for transfection effi ciency with each new batch (
see
Note 1
).
12. 2.5 M CaCl
2
solution: dissolve 36.74 g CaCl
2
· 2H
2
O in 500 mL
of double-distilled water. Filter-sterilize and store in aliquots at
−20 °C. This solution can be frozen and thawed repeatedly.
′
13. Antibiotics for selection:
100× G418 solution: prepare 50 mg/mL active ingredient of
G418 in double-distilled water, fi lter-sterilize, and store at
4 °C protected from light.
Blasticidin S solution conc. 10 mg/mL.
14. Polybrene solution: dissolve 8 mg polybrene in 1 mL sterile
PBS (1,000× stock solution).
15. Crystal violet solution: dissolve 5 mg crystal violet in 125 mL
methanol and 375 mL double-distilled water.
16. Tissue culture media (Dulbecco's modifi ed Eagle's medium
[DMEM], trypsin-ethylene diamine tetraacetic acid solution).
17. BIO-AMF-2 medium (Biological Industries, Kibbutz Beit
Haemek, Israel).
18. For effi cient propagation of primary human amniotic fl uid cells
supplement Ham's F10 medium with 10 % FCS, 1 % Penicillin/
Streptomycin, and 1 % Ultroser™ G (Pall GmbH; cat.no.
15950-017).
3
Methods
3.1 Preparation
of BRK Cells
1. Anesthetize 5-7-day-old neonatal rats, normally a litter of
6-12 pups, by exposure to isofl urane. Alternatively, kill the ani-
mals by cervical dislocation (
see
Note 2
). Place the pups on a
layer of sterile paper towels and remove a section of the skin on
the dorsal side (
see
Fig.
1
). Open the abdominal cavity by an
incision starting from the iliosacral region up to the sternum.
