Supplemental Materials
Expanded Methods
Flow cytometry protocol
To preserve surface antigen expression, Accutase® (Life Technologies) was added to cell
cultures, previously rinsed with PBS, and incubated for 10 minutes. The detached cells were resuspended
in flow cytometry analysis buffer which consisted of 2% (w/v) bovine serum albumin (BSA) and 0.1%
(w/v) sodium azide, diluted in PBS without calcium or magnesium and centrifuged at 200 x g for 5
minutes. The cells were again resuspended in flow cytometry analysis buffer and filtered using a 30-m
filter (CellTrics®, Partec). The cell number was determined using a trypan blue exclusion test (Life
Technologies). To ensure accuracy of antibodies, EPCs were incubated with an Fc receptor blocking
reagent (Biolegend #422301) at a concentration of 5 l per 105 cells prior to primary antibody addition.
Cells were incubated with antibodies for 30 minutes at 40C at a volume of 2 l of antibody per
105 cells with the exception of CD309 (5 l of antibody per 105 cells). The concentrations for each
antibody used was 400 g/ml (CD31 FITC, Biolegend #303103), 50 g/ml (CD45 FITC, Biolegend
#304005), 200 g/ml (CD309/VEGFR2 PE, Biolegend #359903), 200 g/ml (CD115 PE, Biolegend
#347303), 0.2 mg/ml (Mouse IgG1 PE, Biolegend #406607), 100 g/ml (CD146 FITC, Biolegend
#361011), 400 g/ml (CD14 FITC, Biolegend #301803), 25 g/ml (CD34 FITC, Biolegend #343503),
200 g/ml (CD90 FITC, Biolegend #328107), 0.5 mg/ml (Mouse IgG1 FITC, Biolegend #400101), and
200 g/ml (CD105 PE, Biolegend #323205).
Positive controls included human monocytes (THP-1, ATCC) for CD14 and CD45; human
macrophages, induced from THP-1 monocytes, for CD115; human umbilical vein endothelial cells
(HUVECs) (Lonza) for CD31, CD105, CD146, CD309; and human bone marrow-derived mesenchymal
stem cells (MSCs) (Lonza) for CD90. Human monocytes were cultured in RPMI 1640 media containing
glucose, L-glutamine, 10% v/v of FBS, 1% v/v penicillin-streptomycin, and 50 M of 2-mercaptoethanol.
To induce macrophage differentiation, phorbol myristate acetate (Santa Cruz Biotechnology) was added
to the monocyte culture media for a final concentration of 320 nM, and added to monocytes, plated at
1.33 x 104 cells/cm2, for 48 hours. MSCs were cultured in DMEM media containing 4.5 g/L glucose, Lglutamine, sodium pyruvate, 10% v/v FBS and 1% v/v penicillin-streptomycin
After antibody incubation, 200 l of flow cytometry analysis buffer was added and the cells were
centrifuged at 200 x g for 5 minutes. The rinse step was repeated and the cells were preserved through
resuspension in 1% paraformaldehyde (Electron Microscopy Sciences), diluted with PBS.
Fluorescently activated cell sorting (FACS)
To prepare EPCs for cell sorting, samples were prepared using flow cytometry analysis methods
for incubation with CD31 FITC and CD115 PE antibodies. EPCs were sorted at a density of 5 x 105
cells/ml. HUVECs and macrophages served as positive controls for CD31 and CD115, respectively.
Mouse IgG1 conjugated to FITC or PE served as negative controls.