Hla dr clone l243

CD45 clone Hl30 (eBioscience 47–0459-42), CD3e clone OKT3 (eBioscience 46–0037-42), HLA-DR clone L243 (eBioscience 48–9952-42), CD56 clone CMSSB (eBioscience 46–0567-42), CD56 clone HCD65 (BioLegend 318304), CD19 clone H1B19 (eBioscience 46–0198-42 and 56–0199-42), CD14 clone 61D3 (Invitrogen Q10056; Cohort 1), CD14 clone M5E2 (BioLegend 301836; Cohort 2), CD16 clone 3G8 (BioLegend 302040), CD11c clone 3.9 (eBioscience 56–0116-42), CD85g clone 17G10.2 (eBioscience 12–5179-42), BDCA1 clone L161 (BioLegend 331516), BDCA3 clone AD5–14H12 (Miltenyi 130–090-513), CD4 clone RPA-T4 (BioLegend 300550), CD8 clone RPA-T8 (BioLegend 301040), CD25 clone BC96 (BioLegend 302612), CD25 clone 2A3 (eBioscience 17–0259-42), FoxP3 clone PCH101 (eBioscience 77–5776-40), FoxP3 clone 236A/E7 (eBioscience 25–4777-42), PD-1 clone EH-12 (BioLegend 329930), CTLA-4 clone BNI3 (BioLegend 369606), γδ TCR clone B1.1 (BioLegend 331212), Flt3L (Abcam ab9688).

Adherent THP-1 cells were scraped from culture dishes, collected by centrifugation, and resuspended in flow wash buffer (PBS +1% FBS + 0.1% sodium azide). To stain for cell surface markers, cells were incubated in the presence of antibodies specific for galectin 3/Mac-2 (LGALS3, clone M3/38, Cedarlane Labs, Burlington, Ontario, Canada), CCR7/CD197 (clone 3D12), CD206 (clone 19.2), and HLA-DR (clone L243) (eBioscience, San Diego, CA). Cells were then washed and resuspended in 1% paraformaldehyde. Pancreatic cancer cells were collected by trypsinization and resuspended as described above. For MCT4 staining, intracellular staining was performed as previously described (27 (link)) using anti-MCT4 antibody (clone H-90, Santa Cruz Biotechnology) conjugated with Alexa fluor 555 (Z-25305, Life Technologies). Following incubation, cells were washed with 0.1% saponin and then resuspended in flow wash buffer. All samples were collected on an LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (FlowJo, Ashland, OR).