Pd l2 apc

The following Abs were used to stain splenocytes for flow cytometry: B220-BrilliantViolet(BV)605 (BioLegend), Ly5.2-AlexaFluor(AF)700, Ly5.1-PE-Cy7, GL7-APC, CD95-biotin, CCR6-biotin, CD38-FITC, CXCR4-PerCP-eFluor710, CD80-FITC, PD-L2-APC, and Y-Ae-biotin (all from eBioscience). Biotinylated Abs were detected with streptavidin-coupled BV421 (BioLegend). Additional reagents used for flow cytometry included PNA-FITC and NP-PE. Surface staining was carried out as described [29 (link)]. MitoTracker Red (Invitrogen), Annexin V-FITC (eBioscience) and SYTOX Red viability dye (Invitrogen) were used according to manufacturer’s instructions. Data were acquired on a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar, Inc.).

Human monocytes were cultured alone or with mf or three different CMFDA-labeled cancer cell lines (green; FITC channel) as mentioned above. After 48 hrs, cells were harvested, washed with PBS and incubated with 10 μl human IgG (10 mg/ml; Sigma-Aldrich, St. Louis, MO) for 10 min at 4°C to inhibit nonspecific binding through FcγRs and then incubated with marker-specific mAb conjugated with PDL2- APC (eBioscience, Cat No. 17-5888-42), VCAM1(Vascular cell adhesion molecule-1)-APC (Biolegend, Cat No. 305810), CD14-APC Cy7 (eBioscience, Cat No. 47-0149-42), CD45-Pacific blue (Biolegend, Cat No. 304022), CD206 (Mannose receptor)-Percp efluor 710 (eBioscience, Cat No. 46-2069-42), PDL1-PE-Cy7 or PDL1-APC (eBioscience, Cat No. 25-5983-42 and Cat No. 17-5983-41 respectively), or CD163-PE (eBioscience, Cat No. 12-1639-42), CD45-PE (eBioscience, Cat No. 12-9459-42) at saturating concentrations for 30 min at 4°C and washed twice with FACS medium. Monocyte cell populations (CD45⁺/CMFDA^-) were then identified and gated to measure the expression of cell surface markers.