Neutral protease

Whole brain tissue was mechanically and chemically dissociated by Neutral Protease (Worthington), Collagenase Type II (Worthington), Deoxyribonuclease (Worthington), and Complete 1X DMEM (Gibco) with 5% heat inactivated Fetal Bovine Serum (Gibco) and 1% PenStrep (Gibco). 70% Percoll (Cytiva) density gradient was used to separate single cell suspension into cellular fractions. Endothelial cells were labeled with CD31 microbeads (1:10) (mouse, Miltenyi Biotec). Pericytes were labeled with primary antibody rat anti-mouse CD13 (1:50) (Bio-Rad) and secondary anti-rat IgG MicroBeads (1:10) (Miltenyi Biotec). Select cell populations were isolated via magnetic activated cell sorting using LS separation columns (QuadroMACS™ Starting Kit, Miltenyi Biotec). To reduce endothelial cell contamination in the pericyte samples, endothelial cells were removed prior to collecting pericytes. Cell pellets were stored at −80°C until RNA extraction.

The protocol for enzymatic disaggregation of prostate tissue to yield single cells was described previously [20 (link)]. Fresh prostate tissue specimens (1–4 g), or freshly harvested tissue xenograft specimens (pool of ~40 xenografts from 5–8 mouse hosts), were rinsed with PBS, and minced in phenol red-free RPMI medium supplemented with 10% CS-FBS using razor blades. Minced tissue was disaggregated to single cells using a combination of proteases: 1.0 mg/mL Collagenase Type 4 from Worthington Biochemical (Lakewood, NJ) and 0.22 mg/mL Neutral protease from Worthington Biochemical, and incubated at 37°C for 60 minutes. Protease digestion was stopped by low-speed centrifugation of the disaggregation solution contents to pellet the cells and allow removal of the disaggregation buffer supernatant. The pellet was rinsed with HBSS and suspended in 10 mL of DNase buffer (5mM MgCl₂, 5mM CaCl₂, 0.5% BSA) containing 10 mg of DNase from Millipore Sigma (Burlington, MA). After the DNase incubation, the cell suspension was passed sequentially through nylon mesh screens with pores of Falcon 100 μm from Thermo Fisher Scientific (Waltham, MA) and Falcon 40 μm nylon mesh Thermo Fisher Scientific to remove un-disaggregated tissue remnants. The resultant single cell suspension filtrate was used for cell type-enrichment.

Individual capillary endothelial cells were isolated as previously described (Longden et al., 2017 (link)). Brains were denuded of surface vessels with an aCSF-wetted cotton swab, and two 1-mm-thick brain slices were excised and homogenized in ice-cold aCSF using a Dounce homogenizer. The brain homogenate was filtered through a 70 µM filter, and capillary networks that were captured on the filter were transferred to a new tube. Individual capillary endothelial cells were isolated by enzymatic digestion with 0.5 mg/ml neutral protease (Worthington Biochemical Corporation, USA) and 0.5 mg/ml elastase (Worthington Biochemical Corporation) in endothelial cell isolation solution composed of 5.6 mM KCl, 55 mM NaCl, 80 mM sodium glutamate, 2 mM MgCl₂, 0.1 mM CaCl₂, 4 mM glucose, and 10 mM HEPES (pH 7.3; all salts from Sigma-Aldrich, Inc) for 45 min at 37°C. After the first digestion, 0.5 mg/ml collagenase type I (Worthington Biochemical Corporation) was added, and a second 2-min incubation at 37°C was performed. Digested networks were washed in ice-cold endothelial cell isolation solution, then triturated with a fire-polished glass Pasteur pipette to produce individual endothelial cells.

Posterior subcutaneous adipose tissue (gluteal + inguinal) or the brown part of the interscapular fat was dissected, minced with scissors, and digested with 0.1 w.u./ml purified collagenase (LS005273, Worthington Biochemical, Lakewood, NJ, USA) and 2.4 U/ml Neutral Protease (LS02104, Worthington Biochemical) in Hank’s balanced salt solution (HBSS, Sigma-Aldrich, Munich, Germany) containing 4 mM calcium chloride and 0.05 mg/ml DNase I (1284932001, Roche Diagnostics, Grenzach-Wyhlen, Germany) for 50 min at 37°C in a shaker. The suspensions were strained through a 300 μ mesh (4-1411, Neolab, Heidelberg, Germany). Floating mature adipocytes and SVF were separated by centrifugation at 145 × g for 10 min at 20°C. SVF cells were washed, and centrifuged at 300 × g for 5 min at 20°C.