Hello! I want to isolate RBEC from a rat, I used the protocol - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208856/. However, I used 5-day old rats instead of adult rats. Subsequently, the addition of puromycin (4 μg / ml) killed 100% of the cells. Could this be due to differences in the age of the rats, or is it worth looking for other errors?
Attached is a photo of a mixed culture, no puromycin.
Dear Georg!
Please You look at the article and protocol:
Article Puromycin-based purification of rat brain capillary endothel...
Culture of rat brain capillary endothelial cells This method has been adapted from previously described techniques (Roux et al. 1989; Szabo´ et al. 1997; Deli et al. 2000). Briefly, 2-week-old Wistar rat cortices were dissected free of meninges and minced. All animals were treated according to protocols evaluated and approved by the ethical committee of INSERM, France. The dissection was performed on ice and in phosphate-buffered saline (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4,8mM; KH2PO4, 1.5 mM). The cortices were cut into very small pieces (1 mm3 ), homogenized with a 5-mL pipette and digested in a mixture of collagenase/dispase (270 U collagenase/mL, 0.1% dispase) and DNAse (10 U/mL) in DMEM for 1.5 h at 37C. The cell pellet was separated by centrifugation in 20% bovine serum albumin/DMEM (1000 g, 15 min) and incubated in the collagenase/dispase mixture for 1 h at 37C. The capillary fragments were retained on a 10-lm nylon filter, removed from the filter with endothelial cell basal medium supplemented with 20% bovine plasma-derived serum and antibiotics (penicillin, 100 U/mL; streptomycin, 100 lg/mL) and seeded on 60-mm Petri dishes coated with collagen type IV (5 lg/cm2 ). Either 4 lg/mL puromycin was added for 2 days or 3 lg/mL puromycin for 3 days. Puromycin was then removed from the culture medium and replaced by fibroblast growth factor (2 ng/mL) and hydrocortisone (500 ng/mL).
Of course, the more cells are obtained from a younger animal, the more sensitive they are to antibiotics.
Thank you for this protocol. I used lower concentrations: 4-2-1-0.5 μg / ml. At a concentration of 0.5, cell death is weak, but the remaining cells are more similar in morphology to percytes. I think this culture does not contain endothelium. Unfortunately, I cannot hold an ICC at the moment.
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