CD40 Monoclonal Antibody (1C10), APC, eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT. Surface staining was performed in the dark for 30 minutes at 4 °C in staining buffer. Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA).	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. 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. 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).

Protocol tips
cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT. Surface staining was performed in the dark for 30 minutes at 4 °C in staining buffer. Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA).
Downstream tips
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. 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. 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).

Publication protocol

Cell viability was assessed by incubation in the amine-reactive dye Aqua (Invitrogen) (1:500) dilution in Ca2+ and Mg2+ free PBS) for 30 minutes in the dark at room temperature (RT), followed by a single wash in 1x PBS. For all experiments, cells were incubated in 0.5 μg Fc Block (BD Biosciences) for 10 minutes at RT. Surface staining was performed in the dark for 30 minutes at 4 °C in staining buffer. Cells were then washed twice with staining buffer followed by fixation in 1% paraformaldehyde (VWR, West Chester, PA, USA). 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 HL3 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). 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. 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. 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). Fluorescence minus one (FMO) controls were used for gating analyses to distinguish positively from negatively staining cell populations. Compensation was performed using single color controls prepared from BD Comp Beads (BD Biosciences) for cell surface staining or Arc Beads (Invitrogen) for Aqua live/dead discrimination. Compensation matrices were calculated and applied using FlowJo software (Tree Star). Biexponential transformation was adjusted manually when necessary.

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