Live dead near ir dead cell stain

Healthy donor PBMC were thawed, washed and split into four samples. Staining for IgG subtypes was performed in PBS/1% FCS at 4°C in the dark for 15 minutes using the following antibodies and dyes: anti-CD19 V500 (BD Horizon), anti-CD3 APC-Cy7 (BioLegend), anti-CD14 APC-Cy7 (BioLegend), LIVE/DEAD Near-IR Dead Cell Stain (Molecular Probes), anti-CD16 APC-Cy7 (BioLegend), anti-IgD PE-Cy5 (BioLegend, labeled in-house) and either anti-IgG1 PE, anti-IgG2 PE, anti-IgG3 PE or anti-IgG4 PE (all from SouthernBiotech). Cells were washed twice, re-suspended in PBS/1% FCS and cells gated for CD3/14/16/Dead^- CD19⁺ IgD^- and positive for one of the IgG subtypes were sorted on a FACSAriaIII (Becton Dickinson). Sorted cells were frozen at −80°C as dry pellets prior to analysis.

Staining on whole blood was done at room temperature while PBMCs and cells collected from CVL, FS, and CB were stained in PBS containing 2% FBS for 30 min at 4°C. In addition, leukocytes obtained from the first and second CB were separately assayed to compare CD4 T cell yield and phenotype between individual CBs. Cells were stained with fluorochrome-conjugated antibodies specific for CD45 (2D1), CD4 (OKT4), CD8 (SK1), CCR5 (2D7), CXCR4 (12G5), CD27 (M-T271), CD69 (L78) from BD Pharmingen (San Jose, CA); CD45RO (UCHL1), FOXP3 (236A/E7) from eBioscience, (San Diego, CA); CD38 (HIT2) from Invitrogen (Grand Island, NY); and α₄β₇ from the NIH Nonhuman Primate Reagent Resource. Dead cells were excluded from the analysis based on staining for Live/Dead Near-IR dead cell stain from Molecular Probes, Invitrogen. Staining for FOXP3 was performed after cells were stained for surface antigens followed by permeabilization/fixation using the FOXP3 kit and protocol, followed by intracellular staining. Samples were acquired on a LSR Fortessa (BD Biosciences) and all cellular events were collected for mucosal samples while 500,000 events were collected for samples from blood.

U1 snRNP particles were purified from HeLa cells as described before [19 (link)] and SLE-IgG was isolated from two SLE patient sera containing autoantibodies to Sm and RNP autoantigens, by protein G chromatography. The U1 snRNP particles and SLE-IgG were used in cell cultures at final concentrations of 2.5 μg/ml and 1 mg/ml, respectively. All cultures were supplemented with IL-3 (1 ng/ml) and GM-CSF (4 ng/ml) in order to sustain the cell viability of pDCs [20 (link), 21 (link)]. The cell viability of the B cells and pDCs in the 6 day cultures was determined by using LIVE/DEAD near-IR dead cell stain (Molecular Probes, Eugene, OR) and was found to be at least 65–70% of the single cells.