Cd11c pe txr

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.

To evaluate antigen uptake by Ly6C^hi monocytes, MHCII^hi and MHCII^int DCs in the lymph node, Alexa647-labbeled OVA (100 µg/ml) was injected into the footpad of mice mixed with ODN1864 (100 µg/ml). Popliteal lymph nodes were dissected at the indicated time intervals and single cell suspensions were stained with aqua life/dead, Fc block, CD45-MHCII-FITC, Ly6-PE-Cy7, CD11b-APC-Cy7 (BD-biosciences), CD11c-PE-TxR (R & D systems) and Ly6G-PerCP-Cy5.5 (eBioscience). To determine the OVA-Alexa647 positive monocyte/DC gate, mice were injected with a mixture of unlabeled OVA (100 µg/ml) and ODN1864 (100 µg/ml).