Cd11b clone icrf44

The following mAb were used for flow cytometry analysis: CD56 (clone NCAM1), CD16 (clone 3G8) CD3 (clone SK3), CD11b (clone ICRF44), CD14 (clone MOP9) PD1 (clone EH12.1), PD-L1 (clone MIH1), PD-L2 (clone MIH18), CD138 (clone MI15) MICA/B (clone 6D4), CD155 (clone TX24), NKG2A (clone 131,411), NKp44 (clone p44-8), TIM3 (clone 7D3), TIGIT (clone 741,182), DNAM1 (clone DX11) from BD Biosciences; HLA-ABC (clone W6/32), CD38 (clone HIT2), NKp30 (clone p30-15), NKp46 (9E2/NKp46), LAG3 (clone 11C3C65), NKG2D (clone 1D11), CD112 (clone TX31) from BioLegend; CD158a/h/g (clone HP-MA4), CD158e (clone DX9) from Thermofisher, CD158B (clone GL183) from Invitrogen, ULBP256 (clone 165,903) from R&D. All antibodies were titrated prior to usage. Briefly, cells were collected and washed once in PBS. Cells were stained with Aqua live dead cell staining (ThermoFisher) for 20 min at 4 °C, in the dark. Cells were washed once with PBS, containing 2% FBS. Surface staining was performed for 25 min at 4 °C, in the dark. Cells were washed with PBS, containing 2% FBS, centrifuged and fixed with 1% paraformaldehyde for 10 min. Acquisition was performed the following day with Beckman Coulter Cytoflex flow cytometers. Analysis was performed with FlowJo analysis software version 10. Gates were — unless otherwise specified – placed based on the unstained control or FMO (fluorescence minus one).

Cells were surface stained with anti-human antibodies (Abs) to CD11b (clone ICRF44), CD14 (clone TuK4), CD31 (clone M89D3), CD45 (clone 2D1), CD163 (clone GHI/61), IL-6 (clone MQ2-39C3), TNF-α (clone MAb11), vimentin (clone RV202) (BD Pharmingen, San Diego, CA), IL-8 (clone E8N1), CD326 (EpCAM, clone 9C4) (Biolegend, San Diego, CA), and CD3 (clone UCHT1, Beckman-Coulter, Miami, FL). Antibodies conjugated to the following fluorochromes were used in these studies: Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Peridinin chlorophyll protein (PerCP)-Cy5.5, PE-Cy7, Energy Coupled Dye or PE-Texas-Red conjugate (ECD), Pacific Blue, Brilliant Violet (BV) 570, BV605, BV650, Quantum dot (QD) 800, Alexa 647, allophycocyanin (APC)-Alexa 700 and APC-H7.

For flow cytometry analysis, moDCs were harvested, washed, stained, and analyzed by flow cytometry on a Fortessa cytometer (B&D). Live moDCs were defined by sequential gating by forward/side scatter, BV510 viability dye, single cells (SSC-A/SSC-H), and finally CD11b and CD11c (Figure S1). The CD11c⁺/CD11b⁺ population was analyzed for CD80, CD86, CD103, and MHCII, as described in [27 (link)]. The mean fluorescence intensity (MFI) of the aforementioned markers were normalized to the MFI from DCs that were unstimulated. MoDCs matured overnight with LPS (1 µg/mL) and FliC (1 µg/mL) were used as a positive control for staining for surface marker antibodies. The following antibodies were used: CD80 (clone 2D10.4), MHCII/HLA-DR (clone LN3), CD86 (clone IT2.2), CD11c (clone 3.9), and BV506 viability dye (Thermo Fisher), whereas CD103 (clone Ber-ACT8), and CD11b (clone ICRF44) were from B&D. All experiments were performed in triplicate, with three to four different blood donors.