For flow cytometry immunophenotyping experiments, cells were acquired on an LSR II cytometer (BD Immunocytometry Systems, San Jose, CA, USA) equipped with 405 nm, 488 nm, 561 nm, and 640 nm excitation lasers. A comprehensive list of surface markers for these experiments includes: CD45R (B220) clone RA3-6B2 PE-Texas Red (1:250, BD Biosciences), CD4 clone RM4-5 PerCP-Cy5.5 (1:160, BD Biosciences), CD8 clone 53-6.7 PerCP-Cy5.5 (1:160, BD Biosciences), CD8 clone 53-6.7 eFluor 450 (1:333, eBioscience), CD11b clone M1/70 eFluor 450 (1:160, eBioscience, San Diego, CA, USA), CD11b clone M1/70 PE-Texas Red (1:500, Invitrogen (Caltag)), CD11c clone H元 PE-Cy7 (1:125, BD Biosciences), CD16/CD32 clone 2.4G2 PE (1:100, BD Biosciences), CD19 clone 1D3 PerCP-Cy5.5 (1:160, BD Biosciences), CD40 clone 1C10 APC (1:100, eBioscience), CD69 clone H1.2F3 PerCP-Cy5.5 (1:167, BD Biosciences), CD80 clone 16-10A1 PE (1:500, eBioscience), CD86 clone GL1 APC (1:333, eBioscience), CD86 clone GL1 Alexa Fluor 700 (1:100, BD Biosciences), CD115 APC clone AFS98 (1:100, eBioscience), F4/80 clone CI:A3-1 Alexa Fluor 647 (various dilutions, optimized at 1:200, AbD Serotec, Raleigh, NC, USA), F4/80 clone BM8 Alexa Fluor 700 (various dilutions, AbD Serotec), F4/80 clone BM8 APC (various dilutions, optimized at 1:100, eBioscience), F4/80 clone BM8 FITC (various dilutions, eBioscience), F4/80 clone BM8 PE (various dilutions, eBioscience), F4/80 clone BM8 PE-Cy7 (various dilutions, eBioscience), F4/80 clone BM8 PE-Texas Red (various dilutions, eBioscience), F4/80 clone BM8 PerCP-Cy5.5 (various dilutions, eBioscience), Gr-1 clone RB6-8C5 APC-Cy7 (1:160, BD Biosciences), Ly6C clone AL-21 APC-Cy7 (1:500, BD Biosciences), Ly6G clone 1A8 PE (1:416, BD Biosciences), Ly6G clone 1A8 PerCP-Cy5.5 (1:500, BD Biosciences), MHC Class II clone M5/114.15.2 FITC (1:167, eBioscience), MHC Class II clone M5/114.15.2 eFluor 450 (1:500, eBioscience), MHC Class II clone M5/114.15.2 APC-eFluor 780 (1:167, eBioscience), NK1.1 clone PK136 Alexa Fluor 700 (1:160, BD Biosciences), NK1.1 clone PK136 APC (1:286, BD Biosciences), NK1.1 clone PK136 FITC (1:160, BD Biosciences), NK1.1 clone PK136 PerCP-Cy5.5 (1:100, BD Biosciences), mPDCA-1 clone JF05-1C2.4.1 APC (1:10, Miltenyi Biotech), Siglec F clone E50-2440 PE (1:100, BD Biosciences). Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA). Surface staining was performed in the dark for 30 minutes at 4 ☌ in staining buffer. All data collection and sorting were performed using BD FACS Diva software (BD Biosciences) and data analyses were performed using FlowJo software (Tree Star, Ashland, OR, USA).įor all experiments, cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT. The spleen FACS experiments were performed using a FACSAria II instrument (BD Immunocytometry Systems) equipped with 405 nm, 488 nm, or 633 nm lasers located at the University of Chicago Flow Cytometry Core Facility, Chicago, IL, USA. Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA).įor flow cytometry immunophenotyping experiments, cells were acquired on an LSR II cytometer (BD Immunocytometry Systems, San Jose, CA, USA) equipped with 405 nm, 488 nm, 561 nm, and 640 nm excitation lasers. For all experiments, cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT.

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