Flow cytometry and analysis adhered to the general guidelines as previously described[39 (link)]. Single cell suspensions were incubated with Fc block (Tonbo) for 15 minutes at 4°C before staining with surface antibodies and viability dye for 30 minutes at 37°C or 1 hour at room temperature. Antibodies from BioLegend were: CD4 (RM4–5), CD8b (YTS156.7.7), CD25 (PC61), TCRγδ (GL3), NK1.1 (PK136), CD45.2 (104), B220 (RA3–6B2), CD11c (N418), XCR1 (ZET), SIRPα (P84), CD90.2 (30-H12), CD19 (6D5), Ly6G (1A8), CD64 (X54–5/7.1), CX3CR1 (SA011F11), and CD80 (16–10A1). Antibodies from BD Biosciences were: CD8α (53–6.7), TCRβ (H57–597), H2-Kb (AF6–88.5), Siglec F (E50–2440), and CD86 (GL1). Antibodies from eBioscience were: CD5 (53–7.3), PD-1 (J43), CD122 (TM-b1), F4/80 (BM8), MHC Class II (I-A/I-E, clone M5/114.15.2), and Ly6C (HK1.4). CD11b (M1/70) was purchased from Tonbo. Biotinylated CD1d–PBS57 monomers were obtained from the US National Institutes of Health tetramer core and were incubated with APC streptavidin to tetramerize. Samples were acquired on a BD Fortessa or BD LSRII, and data were analyzed with FlowJo 10.
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Detailed Flow Cytometry Protocol
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Antibodies
Cd11b
Cd122
Cd1d
Cd25
Cd90
Cell
Clone
Flow cytometry
Mhc class ii
Siglec
Sirpα
Streptavidin
Surface antibodies
Tcrβ
Tcrγδ
Tetramer
Xcr1
Corresponding Organization : University of Minnesota
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Variable analysis
independent variables
- Fc block incubation time (15 minutes)
- Staining temperature (37°C or room temperature)
- Staining duration (30 minutes or 1 hour)
- Surface marker expression (CD4, CD8b, CD25, TCRγδ, NK1.1, CD45.2, B220, CD11c, XCR1, SIRPα, CD90.2, CD19, Ly6G, CD64, CX3CR1, CD80, CD8α, TCRβ, H2-Kb, Siglec F, CD86, CD5, PD-1, CD122, F4/80, MHC Class II, Ly6C, CD11b, CD1d–PBS57)
- Single cell suspensions
- Antibodies from BioLegend, BD Biosciences, and eBioscience
- Flow cytometry instruments (BD Fortessa and BD LSRII)
- Data analysis software (FlowJo 10)
- Positive control: CD1d–PBS57 monomers tetramerized with APC streptavidin
- Negative control: Not explicitly mentioned
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