Apc conjugated cd34

For FACS analysis, cells were resuspended in a FACS buffer (PBS with 0.1 % BSA and 2.5 mM EDTA). The cell suspension was then stained with PE-conjugated CD43 (Biolegend, clone MEM-59), APC-conjugated CD34 (BD, clone 581) to detect hematopoietic stem/progenitor cells (HSPC). PE-conjugated CD68 (Biolegend, clone Y1/82A), APC-conjugated CD11b (Biolegend, clone ICRF44), FITC-conjugated CD14 (Biolegend, clone HCD14) were used to detect monocyte/macrophages. Basically, cells were incubated with antibodies for 30 minutes at 4°C, followed with washed and suspended in 0.1% BSA/PBS buffer. PE and APC filters were then used to detect cells double positive for CD43 and CD34 or CD68 and CD11b by signal intensity gating, FITC and APC were used to detect cells double positive for CD14 and CD11b. Negative controls stained with control IgG instead of primary antibodies were always performed with sample measurements. Flowcytometry machine of BD FACSAria II and software of Flowjo were mainly used to collect and analyze the flowcytometry data.

Cells were dissociated into single cells with 0.05% trypsin (0.1% EDTA) wherever suitable or rinsed off from culture, and resuspended in a FACS washing buffer (PBS with 5% fetal calf serum (FCS) and 2.5 mM EDTA). The cell suspension was then stained with the desired antibodies. The antibodies used in this study were: CD56 (1:50, clone CMSSB; eBioscience), PE-conjugated CD309 (FLK1, 1:50, clone 7D4–6; Biolegend), PE-conjugated CD13(1:50, clone WM15; BD), FITC-conjugated CD31 (1:50, clone WM59; BD), APC-conjugated CD34 (1:50, clone 581; BD), FITC-conjugated CD43 (1:50, clone MEM-59; Biolegend), PE-conjugated CD43 (1:20, clone eBio84-3C1; eBioscience), FITC-conjugated CD45(1:50, clone 5B1; Miltenyi Biotec), FITC-conjugated CD14 (1:50, clone HCD14; Biolegend), PE-conjugated CD68 (1:50, clone Y1/82A; Biolegend), APC-conjugated CD11b (1:50, clone ICRF44; Biolegend), PE-conjugated CD163 (1:50, clone RM3/1; Biolegend), PE-conjugated CD73 (1:50, clone AD2; Biolegend) and APC/Cy7-conjugated CD163 (1:50, clone 12G5; Biolegend). FITC-conjugated mouse IgG2a (1:20, 130-098-846; Miltenyi Biotec), APC-conjugated mouse IgG1 (1:20, 130-098-877; Miltenyi Biotec) and PE-conjugated mouse IgG1κ (1:20, clone P3.6.2.8.1; eBioscience) were used as isotype-matched negative controls. Data were collected with a FACS Calibur flow cytometer (BD) and analyzed using FlowJo software, version 10.0.7.