FITC Mouse Anti-Human CD70

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	PBMC from patients with autoimmune diseases or from cultures of 5-azaC-treated PBMC from healthy donors were incubated in phosphate-buffered saline (PBS)/0.001% azide containing 10% horse serum at 4°C for 30 min to block non-specific binding. All staining procedures were performed at 4°C in the dark. The cells were then washed, incubated for 60 min, with a single or mixture of Ab.	Fluorescence-activated cell sorting (FACS) analyses were performed using a FACS ARIA IIIU flow cytometer and FACSDiva software V.6.1.3 (Becton Dickinson, New Jersey, USA) or an iCyte Synergy (Sony Biotechnology, San Jose California, USA) and WINLIST V.8 (Verty Software House, http://www.vsh.com) software.

Protocol tips
PBMC from patients with autoimmune diseases or from cultures of 5-azaC-treated PBMC from healthy donors were incubated in phosphate-buffered saline (PBS)/0.001% azide containing 10% horse serum at 4°C for 30 min to block non-specific binding. All staining procedures were performed at 4°C in the dark. The cells were then washed, incubated for 60 min, with a single or mixture of Ab.
Downstream tips
Fluorescence-activated cell sorting (FACS) analyses were performed using a FACS ARIA IIIU flow cytometer and FACSDiva software V.6.1.3 (Becton Dickinson, New Jersey, USA) or an iCyte Synergy (Sony Biotechnology, San Jose California, USA) and WINLIST V.8 (Verty Software House, http://www.vsh.com) software.

Publication protocol

"The following antibodies were used: Phycoerytherin (PE)-anti-Kir2DL4/CD158D (clone 181703; R&D Systems, Minneapolis, Minnesota, USA), anti-CD40L-biotin (clone hCD40L-M91) and avidin-PECy7, PE-Cy7-anti-CD8 (clone RPA-T8), anti-CD11a-APC (clone HI111), Pacific Blue-anti-CD3 (clone UCHT1), PECy5-anti-CD28 (clone CD28.2), FITC-anti-CD70 (clone Ki-24) and APC-Cy7-anti-CD4 (clone RPA-T4) (Becton Dickinson, Franklin Lakes, New Jersey, USA). PE-anti-CD158b (clone CH-L), PE-anti-CD158i (clone FES172), PE-anti-CD158b1/b2, j (clone GL183) and PE-anti-CD158a,h (clone EB6B) were from Beckman Coulter (Brea, California, USA). All antibodies were titrated to determine their optimal concentrations prior to use.

PBMC from patients with autoimmune diseases or from cultures of 5-azaC-treated PBMC from healthy donors were incubated in phosphate-buffered saline (PBS)/0.001% azide containing 10% horse serum at 4°C for 30 min to block non-specific binding. All staining procedures were performed at 4°C in the dark. The cells were then washed, incubated for 60 min, with a single or mixture of fluorochrome-conjugated antibodies. Biotin-CD40L-stained cells were further washed and incubated for 30 min with a 1:1000 dilution of avidin-PE-Cy7, fixed with 4% paraformaldehyde and stored in PBS/0.001% azide in the dark at 4°C until analysed. Controls included isotype and fluorochrome matched antibodies, single antibodies and ‘full minus one’ (FMO) staining controls in which one of each of the fluorochrome-conjugated antibodies was serially omitted while retaining the rest.

Fluorescence-activated cell sorting (FACS) analyses were performed using a FACS ARIA IIIU flow cytometer and FACSDiva software V.6.1.3 (Becton Dickinson, New Jersey, USA) or an iCyte Synergy (Sony Biotechnology, San Jose California, USA) and WINLIST V.8 (Verty Software House, http://www.vsh.com) software. The fluorescent antibody conjugates, laser emission wavelengths and filter sets used are shown in online supplementary table S2."

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