Cd64 bv421

Optimal concentrations of monoclonal antibody combinations directed against the following human surface antigens: HLA-DR-FITC, CD181-FITC (CXCR1), CD86-PE, CD282-PE (TLR2), CD80-PerCP/Cy5.5, CD11b-PerCP/Cy5.5, CD16-PE/Cy7, CD163-PE/Cy7, CD197-APC (CCR7), CD284-APC (TLR4), CD206-APC/Cy7, CD64-BV421, CD14-BV510 (BioLegend) were incubated with THP-1 cells stimulated with IL-1β/IL-1Ra. Isotype matched FITC, PE, PerCP/Cy5.5, PE/Cy7, APC and APC/Cy-7-conjugated irrelevant antibodies (BioLegend) were used as negative controls. Antigen expression was analyzed on a BD FACSCanto II cytometer (BD Biosciences, San Jose, CA). Obtained data were analyzed using FlowJo V10 software (FlowJo, Ashland, OR, USA). In all experiments, a minimum of 10,000 events were counted. Results were expressed as a percentage and median fluorescence intensity (MFI) of the cells for each examined marker.

To analyze monocyte and DC recruitment in response to CpG, 20 µl of a 100 µg/ml ODN1864 solution was injected into the footpad of C57BL/6 J mice. At the indicated time intervals post injection, blood samples were collected through the tail vein followed by ACK (Thermo Fisher Scientific) mediated red blood cell lysis. Alternatively, mice were euthanized and the DLNs were dissected. Single cell suspensions of DLN were prepared through incubation with collagenase type IV (Sigma-Aldrich) and passed through a 70 µm cell strainer (BD Falcon).
After treatment with 2.4G2 Ab for 5 min to block the FcR, PBMCs or lymph node cell suspensions were stained with the following anti-mouse Abs: anti-CD45-V450, anti- MHCII-FITC, anti-CD86-PE, anti-CD40- PE, anti-CD64-APC, anti-F4/80-APC, anti-Ly6C-PE-Cy7, anti-CD11b-APC-Cy7 (all BD Biosciences), Ly6G-PerCP-Cy5.5, CCR2-APC (eBioscience), CD64-BV421 (BioLegend) and CD11c-PE-TxR (R & D systems). Death cells were identified by staining with aqua life/dead stain (Invitrogen). Fluorescent events were acquired using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software. After exclusion of dead cells, granulocytes were identified as CD45+ CD11b+ Ly6C+ Ly6G+ cells and Monocytes as CD45+ Ly6C^hi CD11b^hi Ly6G- cells. Following exclusion of granulocytes and Monocytes in the LN, DCs were subdivided into MHCII^hi DCs and MHCII^int DCs.