Transcription factor fix perm buffer

Synovial cells were resuspended in PBS/1%BSA with primary antibody cocktails at 1:100 dilution for 30 min (Additional file 1: Table S1). All antibodies were obtained from the Longwood Medical Area CyTOF Antibody Resource Core (Boston, MA, USA). Cisplatin was added at 1:400 dilution for the last 5 min of the stain to assess viability. Cells were then washed and fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were then washed and incubated with Ebioscience Transcription Factor Fix/Perm Buffer for 30 min, washed in Ebioscience perm buffer, and stained for intracellular markers at 1:100 for 30 min. Cells were refixed in 1.6% paraformaldehyde for 10 min and stored overnight in PBS/1%BSA. The following day, cells were incubated with MaxPar Intercalator-Ir 500 μM 1:4000 in PBS for 20 min, and then washed twice with MilliQ water, filtered, and analyzed on a Helios instrument (Fluidigm). Mass cytometry data were normalized using EQ™ Four Element Calibration Beads (Fluidigm) as described previously [21 (link)]. viSNE analysis was performed using the Barnes-Hut SNE implementation on Cytobank (www.cytobank.org). Gated live cells (DNA-positive, cisplatin-negative) were analyzed using all available protein markers. Biaxial gating was performed using FlowJo 10.0.7.

We conducted 37 untreated SLE patients and 30 HCs matched for age and gender. Blood samples were stained with fluorophores conjugated antibodies for phenotypic detecting. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation (400 g, 30 min) for cell stimulation. The NK cell subset gating strategies were depicted in Supplementary Fig. 1. All antibodies for staining were shown in Supplementary Table 1. Cell suspensions were surface-labeled with antibodies for 30 min. For intracellular staining, cell suspensions were firstly incubated with surface antibody, then fixed and permeabilized using the Transcription Factor Fix/Perm Buffer (eBioscience). For IFN-γ and IL-10 production assays, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) and ionomycin (1 µg/ml) for 4 h in the presence of Brefeldin A (10 µg/ml). All the cell samples were detected by BD FACSCanto™ II or ACEA NovoCyte and the acquired data were further analyzed with BD FACSDiva software or FlowJo software.