Fitc conjugated mouse igg2a

Cells were dissociated into single cells as described before, and resuspended in a FACS washing buffer (PBS with 5% fetal calf serum (FCS) and 2.5 mM EDTA). To perform direct flow cytometry, the samples were then stained for FITC-conjugated CD31 (1:20; Miltenyi Biotec, Bergisch Gladbach, Germany), and FITC-conjugated mouse IgG2a (for CD31, 1:20; Miltenyi Biotec) was used as isotype-matched negative control. Data were collected with a FACSCaliber flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

For detachment, adherent macrophages were washed with PBS, incubated with 300 µl 10× Gibco™TrypLE™Select (Gibco, Life Technologies) at 37 °C for 15 min and gently collected with a cell scraper after adding 300 µl PBS. Before staining, Fcγ receptor blockage was performed by incubating the cells with 3 mg/ml Beriglobin P (#I4506, Sigma Aldrich) at 4 °C in the dark for 30 min. To determine the purity of isolated primary CD14+ monocytes, cells were stained with either FITC-conjugated mouse anti-human CD14+ REAfinity (#130-110-518, Miltenyi Biotec), FITC-conjugated human IgG1 REAfinity control (#130-113-437, Miltenyi Biotec), PE-conjugated mouse anti-human CD19 monoclonal (clone LT19, #130-091-247, Miltenyi Biotec), PE-conjugated monoclonal isotype control (#120-002-723, Miltenyi Biotec), FITC-conjugated mouse anti-human CD3 monoclonal (#130-080-401, Miltenyi Biotec) and FITC-conjugated mouse IgG2a isotype control (#130-091-837, Miltenyi Biotec) antibodies. To determine the frequency of CD86+ macrophages, cells were stained using anti-CD86-PerCP-eFluor 710 (eBioscience) or isotype control (IgG2b-PerCP-eFluor-710, eBioscience) antibodies. Data were acquired using a BD LSRFortessa™ flow cytometer and analysed using FlowJo software, Treestar Inc.

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.