Cd3 pe cy7

Peripheral blood mononuclear cells were isolated as described before (30 (link), 31 (link)) and stored in liquid nitrogen until use. Briefly, freshly drawn intravenous blood was subjected to ficoll-hypaque gradient centrifugation. 1–2 × 10⁶ PBMCs were first stained with fixable viability dye eFluor 506 (eBiosciences) in order to exclude dead cells from the sample. APC-conjugated PBS-57-loaded CD1d tetramer (NIH Tetramer Core Facility, National Institute of Health, Atlanta, GA, USA) and a panel of following antibodies were purchased from BioLegends; PECy7-CD3, APC-Cy7-CD3, Pacific Blue-CD4, PerCP Cy5.5-CD8, FITC-2B4. Fluorescence-minus-one (FMO) stain was included to avoid false-positive signals. Cells were acquired on a BD FACSCanto II flow cytometer using FACS Diva software (v.7), and analyzed by FlowJo software (v.8.4.4, Tree Star).

Anti-human mAbs included PE-Cy7-CD3 (Biolegend), PerCP-Cy5.5-CD8 (Biolegend), APC-IL-17 (eBioscience), PE-IFN-γ (Biolegend), APC-IL-2 (Biolegend), PE-Cy7-CD19 (Biolegend), FITC-Zombie (Biolegend), APC-fire750-CD69 (Biolegend), PerCP-Cy5.5-TGF-β (Biolegend), APC-IL-10 (Biolegend), PE-GrB (eBioscience). After stimulation, PBMC were harvested and first stained with surface antibodies followed by fixation/permeabilization (Cytofix/Cytoperm kit, BD Biosciences) and subsequent intracellular staining, as previously described [13 (link)].
Flow cytometry was performed in NovoCyte D2060R (ACEA Biosciences Inc). NovoEXpress software (San Diego, CA, USA) was used for analysis. Flow cytometry characterization of lymphocyte subsets is presented in Additional file 1: Figure S1.

Whole human blood was incubated in ACK RBC lysis buffer (Thermo Fisher Scientific) for 4 min at 37 °C to remove red blood cells. Cells were incubated with NIR Zombie (1:200, Biolegend) in PBS for live/dead staining for 15 min at room temperature, followed by BV421 CD45 (1:200, clone 2D1, #368522, Biolegend), APC CD14 (1:200, clone 63D3, #367118, Biolegend), BV605 CD16b (1:200, clone CLB-gran 11.5, #735143 BD), PE/Cy7 CD19 (1:200, clone HIB19, #302216, Biolegend), PE/Cy7 CD3 (1:200, clone HIT3a, #300316, Biolegend) and Fc blocker (1:100, anti-mouse CD16/32,BD) at 4 °C in the dark for 20 min, and washed in PBS/0.5% BSA. Neutrophils, lymphocytes, and monocytes were identified from peripheral blood cells of normal healthy donors with the following high dimensional immunophenotyping; Neutrophils (CD45⁺, CD3/CD19−, CD16b+, CD14⁻), lymphocytes (CD45⁺, CD3/CD19+, CD16b−, CD14⁻) and monocytes (CD45⁺, CD3/CD19−, CD16b−, CD14⁻). Analysis was performed using an LSRII (BD) flow cytometer and FlowJo. v10 (Treestar).

Peripheral blood samples were collected from the included patients around 45 days post-haploHCT. Briefly, 300 μl of fresh peripheral blood per sample was stained with the following fluorochrome-labelled antibodies: PE-Cy7-CD3, BV510-TCRαβ, BV605-CD4, APC-CD8, PE-CF594-CXCR4 (BioLegend, San Diego, CA). After incubation, red blood cells were lysed with a lysis solution (BD Biosciences, San Jose, CA) and then were washed twice with PBS. Polychromatic flow cytometric analyses were performed on a BD LSRFortessaTM Cell Analyser and further analyzed using BD FACSDivaTM software.

PBMCs were thawed and washed twice with PBS. Then, the cells were stained with the following anti-human antibodies: PE-Cy7-CD3, PE/Dazzle594-CD4, APC-Cy7-CD8a, PE-Cy7-CD14, PerCP-Cy5.5-CD16, BV421-CD39, PE-CD73 (BioLegend), anti-nitrotyrosine (anti-NT) rabbit (Cat. #BS-8551R, Bioss Thermo Fisher) and Zombie Aqua (BioLegend). Nitric oxide production was evaluated using the molecular probe DAF-FM DA (10 μM, Cat. #D23844 Invitrogen). The oxidized product was measured at excitation/emission wavelengths of 488/520 nm. Labeled samples were acquired using a BD LSRFortessa FACS cytometer, and data were analyzed using FlowJo™ v10 software (Tree Star, Inc.). The compensation matrixes were designed using UltraComp eBeads™ Compensation Beads (01-222-42; Invitrogen) with specific markers.

Intracellular cytokine staining (ICS) was performed on splenocytes harvested from vaccination-treated BALB/c mice. In detail, two weeks after the second boost immunization, mice in PBS (n=4), RBD/i.n. (n=4), RBD-mFc/i.n. (n=4) and RBD-mFc/i.m. (n=4) were euthanized and spleens were harvested. The splenocytes were seeded into the plates at 1×10⁶ cells/well and stimulated with SARS-CoV-2 RBD protein at 20 μg/mL for 42 hours (37°C, 5% CO₂). Phorbol 12-myristate 13-acetate (PMA, 25 ng/mL) plus ionomycin (500 ng/mL) was used as a positive control, and complete medium alone was used as a negative control. During the last 6 hours, brefeldin A (BFA) was added at 10 μg/mL. Cells were first blocked with TruStain FcX™ PLUS (BioLegend) for 10 minutes then incubated with the following antibodies at 1:200 dilution (BioLegend): PE/Cy7-CD3, PE-CD4, and FITC-CD8. After surface staining, the stained cells were fixed and permeabilized as described in the manufacturer’s instructions (BioLegend) and then stained with APC-IFN-γ or APC-IL-4(1:40, BioLegend). After washing, cell events were acquired using CytoFLEX S (Beckman), and the positive T-cell percentage was analyzed by FlowJo software (FlowJo, LLC).

Thymus was isolated from nondiabetic and diabetic mice perfused with PBS(−) after anesthesia to prepare the suspensions of the thymocyte. Thymocyte suspensions were stained with LIVE/DEAD violet dead cell stain kit (Thermo Fisher Scientific) for 30 min to remove dead cells, washed with PBS(−), and reacted with anti-CD16/32 antibody (Clone 93, Biolegend) to block Fc receptor. Cell suspensions were then stained with PECy7 CD3 (Clone 17A2, Biolegend), FITC CD4 (Clone GK1.5, Biolegend), and APC CD8a (Clone 53-6.7, Biolegend) antibodies for 30 min. The suspensions of stained cells were analyzed using FACS Aria Fusion (BD bioscience). A portion of unstained suspended cells was stained with an isotype control of each fluorescent antibody to determine gating. Acquired data were analyzed using FACS DIVA software (BD Bioscience).