Materials and Reagents

1. 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
      
   
   
2. 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	Nα-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	GibcoTM	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	GibcoTM	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	GibcoTM	21331020	500 ml		4 °C	1x	1x	undiluted
   24	DMEM w/o glucose	Thermo Fisher Scientific	GibcoTM	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	GibcoTM	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

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

Procedure