Quantification of Angiogenic Cell Populations

Basement membrane extract (Trevigen) plugs with growth factors and inhibitors, as indicated, were injected subcutaneously into the flank of nude mice. Single cell suspensions from plugs were prepared as described (24 (link)) and cell populations determined by FACS. Statistics were determined using ANOVA from three independent experiments. Cells were resuspended in PBS, stained with a LIVE/DEAD fluorescent dye (L-23105; Invitrogen), and incubated with CD16/32 (2.4G2; BD Biosciences) to block Fc receptors. For staining, cells were incubated with Alexa Fluor 488-conjugated LYVE-1, PE-Cy7-conjugated CD31, allophycocyanin (APC)-conjugated TER-119, APC-Cy7-conjugated CD45 and eFluor 450-conjugated CD102 (eBioscience or BD Biosciences) washed, and analyzed on a 5-laser LSRFortessa™ (BD Biosciences). Data were analyzed using FACS Diva (BD Biosciences) and FlowJo software (Treestar). Quantification of cell types was performed using PE-conjugated fluorescent counting beads (Spherotech).