Isolating and Culturing Mouse Cerebral Pericytes

Abstract

Source: Mehra, A., et al., A High Output Method to Isolate Cerebral Pericytes from Mouse. J. Vis. Exp. (2020)

This video demonstrates a method for isolating cerebral pericytes from a mouse brain. The isolated cells are cultured in pericyte-specific media to promote pericyte proliferation.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparation of solutions

1. Prepare 500 mL of Washing Buffer A (WBA): 10 mM HEPES solution in Hank's Balanced Salt Solution (HBSS). Store at 4 °C.
2. Prepare 500 mL of Washing Buffer B (WBB): 0.1% Bovine Serum Albumin (BSA) with 10 mM HEPES solution in HBSS. Store at 4 °C.
3. Prepare 300 mL of 30% dextran solution in WBA by mixing the solution overnight at room temperature. Autoclave the solution at 110 °C for 30 min before use. After autoclaving, let the solu....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amino acids BME	Sigma	B-6766	Store at 4 °C.
Basal DMEM media	Invitrogen	316000083	Store at 4 °C.
Basic fibroblast growth factor	Sigma	F-0291	Store at -20 °C.
BSA	Sigma	A-8412	Store at 4 °C.
Collagenase dispase	Sigma	10269638001	Prepare a 10x stock solution in sterile PBS-CMF. Filter the solution with a 0.22 μm syringe filter and store at -20 °C. Note: For the enzyme digestion step of the protocol, for every set of 10 mice for extraction, 300 µL of 10x collagenase dispase is required.
Dextran	Sigma	31398
DNase I	Sigma	11284932001	Prepare a 1000X stock solution by dissolving 100 mg in 10 ml sterile water and store at -20°C.
Gelatin	Sigma	G-2500	Prepare the working coating by making a 0.2% gelatin solution in sterile PBS-CMF (8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, 2.86 g/L NaHPO4 (12 H2O), pH 7.4). Autoclave the solution for minimum 20 minutes at 120 °C and store at room temperature. Culture dishes to be coated for at least 4 hours at 4 °C.
Gentamycin	Biochrom AG	A-2712	Store at 4 °C.
Glutamine	Merck	I.00289	Store at -20 °C.
HBSS	Sigma	H-8264	Store at 4 °C.
HEPES	Sigma	H-0887	Store at 4 °C.
Matrigel	BD Biocoat	354230	Prepare a working coating solution of Matrigel by diluting stock in cold DMEM at 1:48 ratio with its final concentration to be 85 µg/cm2. Cell culture dishes should be coated at least for 1 hour at room temperature.
Pericyte Medium-mouse	Sciencell research laboratories	1231	Store at 4 °C.
Tosyl Lysin Chloromethyl Ketone	Sigma	T-7254	Prepare a 1000X stock solution in WBA by dissolving 16 mg in 10.88 mL of WBA to make a 4 mM solution and store at 4 °C.
Vitamins	Sigma	B-6891	Store at -20 °C.
Filtration tools	Sefar, Nylon mesh, 60-micron porosity
Laboratory equipment	Swing bucket rotor centrifuge
Water bath with agitator
Laminar Flow Hood : BSL2
Glassware (all components to be heat sterilized)	Dounce Tissue Grinder With Glass Pestle
Pestle I: 0.0035 - 0.0065 inches
Pestle II: 0.0010 - 0.0030 inches
Vacuum filter assembly with coarse porosity fritted glass filter support base