Anti cd14 fitc clone m5e2

ADSCs and LDSCs at passages 2, 4, and 6 were analyzed by flow cytometry. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 10⁵ cells/tube), washed with FACS buffer (PBS containing 2% FBS), and then incubated with the following fluorescein (FITC)- or phycoerythrin (PE)-conjugated antibodies: anti-CD14-FITC (clone M5E2; BD Pharmingen), anti-CD29-PE (clone TS2/16; BioLegend), anti-CD34-PE (clone 1H6; R&D Systems), anti-CD44-PE (clone IM7; BioLegend), anti-CD45-FITC (clone YKIX716.13; eBioscience), and anti-CD90-PE (clone YKIX337.217; eBioscience) or their respective isotype controls [11 (link), 12 (link)]. The cells were washed twice with FACS buffer and resuspended in 500 μl FACS buffer. Fluorescence was evaluated by flow cytometry using a FACSCalibur instrument (BD Biosciences). Data were analyzed using WinMDI 2.9 analysis software.

Peripheral whole blood samples were collected in sterile BD Vacutainer^® EDTA (ethylenediamine-tetraacetic acid) blood collection tubes (BD Biosciences). The samples were incubated in BD Trucount™ tubes (BD Biosciences) with the following monoclonal antibodies to identify lymphocyte sub-populations (BD Biosciences): anti-CD45-PerCP (clone 2D1), anti-CD3-FITC (clone UCHT-1), anti-CD4-APC (clone RPA-T4), anti-CD8-PE (clone RPA-T8), anti-CD16-PE (clone 3G8), anti-CD19-APC (clone HIB19), anti-CD56-PE (clone NCAM16.2). The following antibodies were used for monocyte sub-populations (BD Biosciences): anti-CD45-APC (clone 2D1), anti-HLA-DR-PerCP (clone L243), anti-CD14-FITC (clone M5E2), and anti-CD16-PE (clone 3G8). The samples were incubated at 4 °C for 30 min and then were treated with FACS Lysing Solution (BD Biosciences) until the erythrocytes were lysed and the cells were immediately processed in the FACSCanto flow cytometer (Becton–Dickinson Immunocytometry Systems, Palo Alto, CA) along with 10,000 beads per tube. The results were analyzed with FACSDiva Software (BD Biosciences) and the absolute numbers of lymphocytes and monocytes subsets were calculated on the basis of bead counts. Monocyte populations were classified as classical (CD14++ CD16−), intermediate (CD14++ CD16+), and non-classical (CD14+ CD16++).12 (link)