PE-Cy™7 Rat anti-Mouse CD117

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Samples were isolated from mouse tissues or tumors as above or from trypsinized from tissue culture samples.	Unstained samples and samples with antibody IgG controls were run in parallel. Additionally, color compensation for each fluorochrome was evaluated using IgG compensation beads (552843, BD Pharmingen) and ArC compensation beads (A10346, Invitrogen) with appropriate fluorescent marker controls.	Flow cytometry was performed in the Stanford Flow Cytometry Core Facility using the BD Aria II (sorting) or BD LSR.II (analysis).

Upstream tips
Samples were isolated from mouse tissues or tumors as above or from trypsinized from tissue culture samples.
Protocol tips
Unstained samples and samples with antibody IgG controls were run in parallel. Additionally, color compensation for each fluorochrome was evaluated using IgG compensation beads (552843, BD Pharmingen) and ArC compensation beads (A10346, Invitrogen) with appropriate fluorescent marker controls.
Downstream tips
Flow cytometry was performed in the Stanford Flow Cytometry Core Facility using the BD Aria II (sorting) or BD LSR.II (analysis).

Publication protocol

Flow cytometry was performed in the Stanford Flow Cytometry Core Facility using the BD Aria II (sorting) or BD LSR.II (analysis). Samples were isolated from mouse tissues or tumors as above or from trypsinized from tissue culture samples. Cells were blocked with CD16/CD32 Mouse Fc Block (553142, BD Pharmingen) for 30 minutes, then labeled with anti-Flk-1:PE, anti-c-Kit:PE-Cy7, anti-CD45: APC-Cy7, and as indicated, anti-CD31: FITC. Unstained samples and samples with antibody IgG controls were run in parallel. Additionally, color compensation for each fluorochrome was evaluated using IgG compensation beads (552843, BD Pharmingen) and ArC compensation beads (A10346, Invitrogen) with appropriate fluorescent marker controls. Once lasers were tuned and color compensation was set, at least 100,000 cells per sample were run, except for the adipose tissue samples which were run at 50,000 cells per sample. Gating was initially applied to isolate single cell populations of viable cells, then additional gating was used to select for the appropriate subpopulations (Flk-1+/c-Kit+/CD45-). Flow cytometric analysis was performed using FlowJo Software (TreeStar, Inc., Ashland, OR). For the flow cytometry sorting of live cells, the same staining process was used as above. Once subpopulations of single, viable cells were gated on Flk1+, c-Kit+, and CD45- and subsequently isolated, they were then collected in an eppendorf tube with 500 μL of mouse endothelial media (M1168, Cell Biologics, Chicago, IL) supplemented with the growth factor kit (VEGF, EGF, heparin, hydrocortisone, and L-Glumatine) and 10% FBS and managed as in vitro cultures as described below.

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