Apc cy7 anti human cd3

From peripheral blood samples collected from 21 pSS patients and 20 CS, peripheral blood mononuclear cells (PBMC) were obtained by Ficoll density-gradient centrifugation. PBMC were washed with phosphate-buffered saline. Their viability was evaluated using the trypan blue exclusion method, and only those samples with more than 90% of viability were considered. Multicolor flow cytometry was used to analyze from PBMC. The expression of ICOS on gated CD4+, and CD3+ T cells was analyzed. Cell surface staining was performed with fluorochrome-labeled monoclonal primary antibodies purchased from Biolegend: APC-Cy7 anti-human CD3 (cat: 300426), AF488 anti-human CD4 (cat: 300518), and AF700 anti-human CD278/ICOS (cat: 313528). Corresponding isotype control antibodies were also included from Biolegend. Assay tubes were stained with a mixture of corresponding antibodies at the recommended dilution and incubated for 30 min at room temperature in the dark. After incubation, the cells were washed and fixed. Finally, data were acquired using Attune NxT (Thermo Scientific, Waltham, MA, USA). Data were analyzed with FlowJo software version 10.7 (Becton Dickinson Biosciences Company, NJ, USA).

The B-cell count was determined by immunofluorescence staining and flow cytometric analysis using following antibodies from BioLegend: Pacific Blue-anti-CD45 (Cat#304029), APC/Cy7-anti-human CD3 (Cat#300426), PE-anti-CD19 (Cat#302208), FITC-anti-CD20 (Cat#302304), PE/Cy7-anti-human Ig light chain κ (Cat#316520), and APC-anti-human Ig light chain λ (Cat#316610). Live cells were determined by 7-AAD staining, and normal B-lineage cells were defined as CD45⁺CD3^–CD19⁺CD20⁺κ⁺λ⁺ lymphocytes. The normal range for blood B cells is 61–321 cells/μL^{35 (link)}.

Cryo-preserved PBMCs of healthy volunteers or HNSCC patients and corresponding disintegrated primary tumor samples were stained with a cocktail of mouse anti-human CD3-APC.Cy7 (clone HIT3a), anti-human CD4-PacificBlue (clone OKT4), anti-human CD8a-AlexaFluor700 (clone HIT8a), anti-human CD16-FITC (clone 3G8), anti-human CD56-PE.Cy7 (clone HCD56) (all, BioLegend, USA), anti-human CD45-Pe.Flour610 (clone HI30; eBioscience, USA), and anti-human CD314-PE (clone BAT221; Miltenyi Biotec, Germany). For cell viability, Aqua Dead Cells stain (Thermo Fisher Scientific, USA) was used. Cellular events were measured on a Gallios cytometer and analyzed using Kaluza software (both Beckmann Coulter, USA). Percentage of NK cell subsets and mean fluorescent intensity ratios for NKG2D expression were calculated from CD45⁺ lymphocyte fraction.

PBMC were harvested from Colon Intestine-Chip epithelial channels at the final timepoint after administration. PBMC were washed with DPBS and stained with 2 μM live/dead Fixable Yellow Dead Cell Stain (ThermoFisher) and washed in DPBS. PBMC were then fixed with BD Cytofix (BD Biosciences) fixation solution, washed in DPBS, and either resuspended in 90% FBS (Sigma) + 10% DMSO solution and frozen at −20°C until use or stained immediately. Samples to be stained for surface markers were washed in DPBS and resuspended in Cell Staining Buffer (Biolegend). Surface marker stains were prepared in BD /Cytoperm solution (BD Biosciences) and consisted of anti-human CD3 APC-Cy7 (BioLegend), anti-human CD4 Brilliant Violet 786 (BioLegend), anti-human-CD8-PE/Dazzle-594 (BioLegend), and anti-human CD69 APC (BioLegend).
Sample data was acquired using the BD FACSCelesta flow cytometer (BD Biosciences), and data was analyzed using FlowJo V10 software (FlowJo).