Anti hla dr apc cy7

Peripheral blood mononuclear cells from healthy donors (Hoxworth blood center, Cincinnati, OH) were separated by centrifugation through Ficoll-Hypaque (GE, Fairfield, CT). CD4+ T cells were purified by negative selection using the Miltenyi CD4 negative separation kit (Auburn, CA), per the manufacturer’s instructions. Aliquots of purified CD4+ T cells were frozen in FBS + 10% DMSO and stored in liquid nitrogen. The viability of thawed cryopreserved cells was >90%. To isolate bulk Treg, cryopreserved CD4+ T cells were stained with live dead aqua (ThermoFisher, Waltham, MA), anti-CD8-FITC, anti-CD25-APC (BD Pharmingen, San Diego, CA), anti-CD127-PE (Beckman Coulter, Fullerton, CA), and sorted using a FACS Aria (BD) (supplemental Fig. S1A). Purity of the sorted Treg was >90%, as determined by postsorting analysis of FOXP3 expression (supplemental Fig. S1B). FOXP3 expression was also significantly higher in Treg compared with conventional CD4+T cells (non-Treg) after sorting (supplemental Fig. S1C). In some experiments, Treg subsets (nTreg, mTreg, and emTreg) were isolated, briefly cryopreserved CD4+ T cells were stained with anti-CD8-FITC, anti-CD25-APC, anti-HLA-DR-APC-Cy7 (BD), anti-CD127-PE (Beckman Coulter), anti-CD45RA-PB, anti-CD95-BV510 (BioLegend, San Diego, CA), and sorted using an FACS Aria (supplemental Fig. S1A).

PBMCs were stained with the following list of fluorescently labeled antibodies against cell surface markers subsequently detected by flow cytometry: anti-CD14 BV605 (clone: M5E2), anti-CD16 PE-Cy7/BV711 (clone: 3G8), and anti-CD3 PE (clone: HIT3a) from BioLegend; anti-CD20 e450 (clone: 2H7), anti-CD19 e450 (clone: SJ25C1), anti-CD2 e450 (clone: RPA-2.10), and anti-IL-1β (clone: CRM56) from eBioscience; anti-CD56 e450 [clone: B159 (RUO)], anti-HLA-DR APC-Cy7 [clone: G46-6 (RUO)], and anti-CD66b e450 (clone: G10F5) and CCR2 PerCP Cy5.5 from BD Biosciences. Staining protocol proceeded as described above for imaging flow cytometry analysis. Surface Glut-1 receptor expression was detected by binding to the Glut-1 ligand fused to enhanced green fluorescent protein (GFP) (Metafora Biosystems, Evry, France), as previously described (53 (link)). In brief, 10⁶ cells were incubated for 20 min with 10 µl of Glut-1–GFP solution prior to Live/Dead staining using LIVE/DEAD Cell Viability Assay (Life Technologies, CA, USA). GFP fluorescence was detected by staining cells with Alexa Fluor 647 anti-GFP antibody at 1:200 (Biolegend). Data were acquired on a BD Fortessa flow cytometer (BD Biosciences). All compensation and gating analyses were performed using FlowJo 10.5.3 (TreeStar, Ashland, OR, USA).