FITC Mouse Anti-Mouse NK-1.1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	From the blood, kidney, brain, spleen, and liver single-cell suspensions, 500 μl each were stained with a Live/Dead fluorescent dye (Invitrogen, Carlsbad, Calif., USA) for 10 min (1:500) and then incubated with rat anti-mouse CD16/32 (2.4G2; BD Biosciences) for 10 min (1:100) at 4°C to block Fc receptors	After three washes with FACS buffer, cells were fixed with 2% paraformaldehyde. FACS was performed on an LSRII (BD Biosciences) and data were analyzed using FlowJo software (version 8.8.4; Treestar, Ashland, Oreg., USA).

Protocol tips
From the blood, kidney, brain, spleen, and liver single-cell suspensions, 500 μl each were stained with a Live/Dead fluorescent dye (Invitrogen, Carlsbad, Calif., USA) for 10 min (1:500) and then incubated with rat anti-mouse CD16/32 (2.4G2; BD Biosciences) for 10 min (1:100) at 4°C to block Fc receptors
Downstream tips
After three washes with FACS buffer, cells were fixed with 2% paraformaldehyde. FACS was performed on an LSRII (BD Biosciences) and data were analyzed using FlowJo software (version 8.8.4; Treestar, Ashland, Oreg., USA).

Publication protocol

From the blood, kidney, brain, spleen, and liver single-cell suspensions, 500 μl each were stained with a Live/Dead fluorescent dye (Invitrogen, Carlsbad, Calif., USA) for 10 min (1:500) and then incubated with rat anti-mouse CD16/32 (2.4G2; BD Biosciences) for 10 min (1:100) at 4°C to block Fc receptors. Then, cells were incubated at 4°C for 30 min with the following antibodies: FITC-conjugated anti-mouse Ly6c (AL-21), CD3 (17A2), NK1.1 (PK136; BD Biosciences), and MHC II (M5/114.15.2; eBioscience, San Diego, Calif., USA); PE-conjugated anti-mouse CD45 (Ly-5) and γδ T-cell receptor (GL3; BD Biosciences); PE-Cy7-conjugated anti-mouse CD45 (Ly-5; BD Biosciences), CD8 (53-6.7), and F4/80 (BM8; eBioscience); APC-conjugated anti-mouse CD45 (Ly-5), CD11c (HL3; BD Biosciences), and CD19 (1D3; eBioscience); APC-Cy7-conjugated anti-mouse CD11b (M1/70) and CD3 (17A2; BD Biosciences), and APC-fluor-780-conjugated anti-mouse CD4 (RM4-5; eBioscience). After three washes with FACS buffer, cells were fixed with 2% paraformaldehyde. FACS was performed on an LSRII (BD Biosciences) and data were analyzed using FlowJo software (version 8.8.4; Treestar, Ashland, Oreg., USA).

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