从成年小鼠分离和培养原代脑内皮细胞

Julian Christopher Assmann; Kristin Müller; Jan Wenzel; Thomas Walther; Josefine Brands; Peter Thornton; Stuart M. Allan; Markus Schwaninger

doi:10.21769/BioProtoc.2294

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Peer-reviewed

Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

从成年小鼠分离和培养原代脑内皮细胞

JA Julian Christopher Assmann

MS Markus Schwaninger email

发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2294 浏览次数: 14442

评审: Oneil G. BhalalaAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Sep 2016

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Abstract

Brain endothelial cells are the major building block of the blood-brain barrier. To study the role of brain endothelial cells in vitro, the isolation of primary cells is of critical value. Here, we describe a protocol in which vessel fragments are isolated from adult mice. After density centrifugation and mild digestion of the fragments, outgrowing endothelial cells are selected by puromycin treatment and grown to confluence within one week.

Keywords: Primary culture (原代培养)

Blood-brain barrier (血脑屏障)

Tight junctions (紧密连接)

Background

The blood-brain barrier protects the brain from uncontrolled entry of cells and substances. This is mainly achieved by brain endothelial cells that form a barrier composed of tight and adherens junctions to restrict paracellular transport.

This protocol was developed to overcome the limited availability of mouse brain endothelial cell lines that maintain their key characteristics, e.g., the expression of sufficient amounts of tight junction proteins such as occludin, ZO-1 or claudin-5 to induce a high transendothelial resistance.

In addition, the isolation of brain endothelial cells from genetically modified mice allows investigating of gene-specific functions in vitro.

Using this method, we previously complemented in vivo studies demonstrating the importance of NF-κB signaling in brain endothelial cells for maintaining normal blood-brain barrier function (Ridder et al., 2015).

Materials and Reagents

Materials
1. Multiwell plate (cell culture grade) (Greiner Bio One International, catalog number: 662160 )
2. Cellulose chromatography paper (sterilize at 180 °C) (Whatman, catalog number: 3030-931 )
3. 50 ml centrifuge tubes (cell culture grade) (Greiner Bio One International, catalog number: 210261 )
4. 10 ml disposable pipette (Greiner Bio One International, catalog number: 607160 )
5. Mice (C57BL/6, age 6 weeks up to 1 year from Charles River, Germany or the in-house breeding facility)
6. Ice

Reagents

	Reagents	Manufacturer	Brand	Catalog number	Preparation	Aliquots	Storage	Stock concentration	Working concentration	Dilution
1	Hydrochloric acid (HCl) 1 N (sterile filtered)	Carl Roth		K025.1	1 L		RT		0.05 N	1 ml HCl 1 N + 19 ml H₂O, sterile
2	Dulbecco’s PBS (DPBS)	Biowest		L0615-500	500 ml		4 °C	1x	1x	undiluted
3	70% EtOH (denatured)	Th. Geyer		2270			RT
4	Isoflurane	Baxter		KDG9623			RT
5	Collagenase/dispase (sterile filtered)	Roche Diagnostics		11097113001	500 mg/5 ml H₂O, sterile	200 µl	-20 °C	100 mg/ml	1 mg/ml	100 µl collagenase/dispase in 10 ml medium
6	DNase I	Roche Diagnostics		11284932001	100 mg/10 ml H₂O, sterile	100 µl	-20 °C	10 mg/ml	4 µg/ml	40 µl DNase I in 10 ml medium
7	N_α-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK)	Sigma-Aldrich		90182	14.7 mg/10 ml H₂O, sterile	200 µl	-20°C	1.47 mg/ml diluted to 14.7 µg/ml	0.147 µg/ml	100 µl TLCK in 10 ml medium
8	Puromycin	Sigma-Aldrich		P8833	2.5 mg/10 ml H₂O, sterile	500µl	-20°C	0.25 mg/ml	8 µg/ml	32 µl puromycin in 1 ml medium
9	Trypsin-EDTA 0.25%	Thermo Fisher Scientific	Gibco^TM	25200-056	100 ml		-20°C	1x	1x	undiluted
10	4% paraformaldehyde	Merck Millipore		1040051000	4% paraformaldehyde in DPBS		-20°C
11	CD31	BD	BD Pharmingen	557355						1:500
12	α-smooth muscle Actin (α-SMA)	Acris Antibodies		DM001-05
13	Iba1	Wako Pure Chemical Industries		019-19741
14	Glial Fibrillary Acidic Protein (GFAP)	EMD Millipore		AB5541
15	Zona occludens-1 (ZO-1)	Thermo Fisher Scientific	Invitrogen	40-2200
16	VE-Cadherin	Santa Cruz Biotechnology		sc-6458
17	Claudin-5 (Cldn-5)	Thermo Fisher Scientific	Invitrogen	34-1600
18	Occludin (Ocln)	Sigma-Aldrich		SAB3500301
19	Mouse collagen, type IV	Corning		354233	890 mg/ml 0.05 N HCl	100 µl	-80 °C	890 mg/ml	50 µg/ml	56 µl collagen IV + 944 µl 0.05 N HCl
20	Dextran MW 60,000-90,000	Alfa Aesar		J14495	1 kg		RT		18%	5.4 g dextran in 30 ml DPBS
21	Penicillin/streptomycin (100x)	Biochrom		A2212	100 ml	1 ml	-20 °C	100x	1x	100 µl pen/strep in 10 ml medium/dextran
22	L-glutamine	Thermo Fisher Scientific	Gibco^TM	25030024	100 ml	1 ml	-20 °C	200 mM (100x)	2 mM (1x)	100 µl L-glutamine in 10 ml medium
23	DMEM-F12 w/o glutamine	Thermo Fisher Scientific	Gibco^TM	21331020	500 ml		4 °C	1x	1x	undiluted
24	DMEM w/o glucose	Thermo Fisher Scientific	Gibco^TM	11966025	500 ml		4 °C	1x	1x	undiluted
25	Plasma-derived bovine serum (PDS)	First Link		60-00-810	500 ml	10 ml	-20 °C	100%	20%	10 ml PDS in 50 ml medium
26	Antibiotic/antimycotic (100x)	Thermo Fisher Scientific	Gibco^TM	15240062	100 ml	1 ml	-20 °C	100x	1x	100 µl AA in 10 ml medium/dextran
27	Heparin-sodium	Ratiopharm		PZN 003029843	1 ampule		4 °C	5,000 I.E./ml	750 I.E./50 ml	150 µl heparin in 50 ml medium
28	Endothelial Cell Growth Supplement (ECGS)	Sigma-Aldrich		E2759	15 mg/5 ml DPBS	500 µl	-20 °C	3 mg/ml	30 µg/ml	500 µl ECGS in 50 ml medium
29	18% dextran solution (see Recipes)
30	Working medium (see Recipes)
31	Digestion medium (see Recipes)
32	Full medium (see Recipes)

Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight. Vortex thoroughly. Aliquot and store at -80 °C. The collagen concentration varies from lot to lot. Therefore, the amount of HCl added has to be adjusted for every new lot.

Equipment

Refrigerator (4 °C)
Shaker
Sterile beakers 100-150 ml (sterilize at 180 °C)
Laminar flow work bench
Dounce tissue grinder, 15 ml, autoclave (Sigma-Aldrich, catalog number: D9938 )
Scalpel
Tweezers (sterilize at 180 °C)
Centrifuge (Hettich Lab Technology, model: UNIVERSAL 320 R ),
Fixed-angle rotor (Hettich Lab Technology, catalog number: 1620A )
Big scissors (sterilize at 180 °C)
Small scissors (sterilize at 180 °C)
Pipette or vacuum pump
Water bath
Microwave oven

Procedure

English

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文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Assmann, J. C., Müller, K., Wenzel, J., Walther, T., Brands, J., Thornton, P., Allan, S. M. and Schwaninger, M. (2017). Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice. Bio-protocol 7(10): e2294. DOI: 10.21769/BioProtoc.2294.
Khan, M. A., Schultz, S., Othman, A., Fleming, T., Lebron-Galan, R., Rades, D., Clemente, D., Nawroth, P. P. and Schwaninger, M. (2016). Hyperglycemia in Stroke Impairs Polarization of Monocytes/Macrophages to a Protective Noninflammatory Cell Type. J Neurosci 36(36): 9313-9325.