Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination
Published: April 30, 2023
Abstract
Source: Hørlyck, S. et al. Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies. J. Vis. Exp. (2020)
In this video, we demonstrate the procedure to isolate and culture pericytes from bovine brain capillaries without endothelial cell contamination.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Seeding and Culturing of Bovine Capillaries Day 0: Mix 0.7 mL of collagen IV stock with 6.3 mL of PBS. Add the solution to a T75-flask and leave the flask for 2 h at room temperature (RT) or leave it overnight at 4 °C. Remove the collagen solution from the flask and wash three times …
Representative Results
Figure 1: Culturing of capillaries and isolation of pericytes. Capillaries have been isolated from fresh bovine brain and seeded in culture flasks on day 0. Outgrowth from the bovine brain capillaries and the following isolation of pericytes were followed over days with a light microscope. Day 4a shows the endothelial cell growth prior to treatment with trypsin to remove endothelial cells an…
Açıklamalar
The authors have nothing to disclose.
Materials
| PBS | Sigma Aldrich | D8537 | Phosphate-buffered saline |
| Collagen IV | Sigma Aldrich | C5533 | |
| T-75 flask | Sigma Aldrich | CLS3972 | |
| Fetal bovine serum (FBS) | PAA/GE Healthcare | A15-101 | |
| Fibronectin | Sigma Aldrich | F1141 | |
| Trypsin-EDTA | Sigma Aldrich | T4299 | |
| Light microscope | Olympus | Olympus CK2 | Upright light microscope with phase contrast |
| DMSO | Sigma Aldrich | 471267 | |
| Heparin | Sigma Aldrich | H3149 | |
| Cell incubator | Thermo Fisher | ||
| Centrifuge | Thermo Fisher | Heraeus Multifuge 3SR+ | Standard large volume centrifuge for spinning down cells |
| Dulbecco's Modified Eagles Medium | Sigma Aldrich | D0819 | |
| Penicillin G Sodium/Streptomycin sulfate | Sigma Aldrich | P0781 | |
| MEM nonessential amino acids | Sigma Aldrich | M7145 |
Etiketler
Bu Makaleden Alıntı Yapın
Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination. J. Vis. Exp. (Pending Publication), e20681, doi: (2023).