Collagenase

Human adipose lipoaspirates were obtained after informed consent from female patients (26–54 years old). Fresh lipoaspirates were separated from blood and lipoaspiration fluid were passed through a 100 μm nylon mesh. Solid matter was then resuspended in collagenase digest buffer (2.2mg/ml collagenase II (Sigma) supplemented with 100U/ml DNase (Worthington) in M199 Media (Sigma)), shaken in CERTOMAT^® BS-1 Incubation-Shaking Cabinet at 37°C for 30 minutes, sedimented on Histopaque-1119 Density gradient to remove blood and dead cells, washed, and pelleted at 400 x g at 4°C. Cells were then plated on Lonza Endothelial Growth Media and transduced with CMV-GFP expressing lentivirus. Twenty-four hours after transduction, cells were lifted with collagenase, resuspended in staining media (2% FBS in PBS), blocked with rat IgG and stained with fluorochrome-conjugated antibodies against human CD45, Tie2, PDGFRα, and CD31 for FACS on the BD Aria II Flow Cytometer. As with mouse samples, the gating was adjusted based on compensation, FMO controls and, unstained cells. In ischemic rescue studies, 500,000 sorted VSPC1+VSPC2 vessel-forming progenitors were injected around ligated femoral artery of 8–12 weeks old immunodeficient Rag2/gamma(c) knockout male mice.

A protocol for primary hepatocyte isolation was performed using Collagenase (BD Biosciences) and gradient centrifugation as adapted from previously published methods (Berry and Friend, 1969 (link); Li et al., 2010 (link)).

Isolation of CD8⁺ T cells was performed similarly as described^{31 (link)}. For isolation of CD8⁺ T cells from the small intestine intraepithelial lymphocyte (IEL) compartment, Peyer’s patches were removed and the intestine was cut longitudinally and subsequently cut laterally into 0.5–1cm² pieces that were then incubated with 0.154mg/mL dithioerythritol (DTE) in 10% HBSS/HEPES bicarbonate for 30min at 37°C while stirring. Kidneys, salivary glands, and lungs were cut into pieces and digested for 30min with 100 U/mL type I collagenase (Worthington) in RPMI 1640, 5% FBS, 2mM MgCl₂, 2mM CaCl₂ at 37°C while shaking. Skin was processed similarly as described^{32 (link)} in which a 2cm² area of the right flank was excised, pre-digested for 30min at 37°C and then enzymatically digested with 0.7 mg/mL collagenase D. After enzymatic incubations (skin, lungs, kidneys, salivary glands), tissues were further dissociated over a 70μm nylon cell strainer (Falcon). For isolation of lymphocytes, single-cell suspensions were then separated using a 44/67% Percoll density gradient. Spleens and lymph nodes were processed with the frosted ends of microscope slides. Red blood cells were lysed with ACK buffer (140 mM NH₄Cl and 17 mM Tris-base, pH 7.4).