Rat igg1 apc

Astrocytes from four controls and six BD patients were differentiated for 5 weeks and stimulated with 10 ng/mL recombinant human IL-1β (R&D) or with 50 ng/mL recombinant human TNF-α (R&D) or PBS (vehicle) for 5 h. Protein transport inhibitors (1:1,000 BD GolgiPlug and BD GolgiStop; BD Biosciences) were added to all samples, including non-stimulated controls. Five hours after stimulation, cells were dissociated at room temperature for 1 min in a 1:1 mixture of enzymes accutase (STEMCELL) and papain (Papain dissociation system, Worthington). Cells were washed and stained with the viability dye Zombie UV fixable kit (BioLegend). Cells were fixed and permeabilized using the BD Cytofix/Cytoperm and BD Perm/Wash (BD Biosciences). IL-6 or IL-8 cytokines were detected following incubation in BD Perm/Wash containing PerCP conjugated anti-IL-8 (BioLegend BH0814) and APC conjugated anti-IL-6 (BioLegend MQ213A5) antibodies for 20 min at 4°C. Quantification was done on the BD Canto II cytometer (BD Biosciences) and analysis was performed on FlowJo software (TreeStar). Negative gating controls for anti-IL-6 were done with rat IgG1-APC (BioLegend RTK2071) and for anti-IL-8 with mouse IgG2b-PercP (BioLegend MPC-11). Also, negative gates were determined for each experiment using non-stimulated controls, and normalized by subtraction from positive cells (data not shown).

Human cells were stained with the following fluorochrome-labeled antibodies: anti-CD45-PECy7 (Biolegend), anti-CD8-BV510 (Biolegend), anti-CD25-BV421 (Biolegend), anti-CD69-PerCPCy5.5 (Biolegend), anti-CD137-Biot (5D1, the laboratory’s own hybridoma), anti-Ki67-AF488 (Biolegend) and anti-Granzyme-AF647 (Biolegend). Streptavidin-PE was added to detect 5D1-Biot Ab. Isotype control mixes was prepared with mIgG1-BV421 (Biolegend), mIgG1-PerCPCy5.5 (Biolegend), mIgG1-AF488 (Biolegend), mIgG1-AF647 (Biolegend). In the case of 5D1-Biot, FMO was performed. M11 CAR expression was verified with anti-mIgG(H+L)-AF647 (Invitrogen) just before stimulation with mesothelin-Fc-beads.
Mouse T cells were stained with: anti-CD8-PECy7 (Biolegend), anti-CD25-APC (Biolegend), anti-CD69-BV510 (Biolegend), anti-CD137-PE (Biolegend), anti-PD1-PerCPCy5.5 (Biolegend), and anti-Ki67-AF488 (BD Bioscience). Rat IgG1-APC (Biolegend), Arm Hamster-BV510 (Biolegend), Syr Hamster-PE (Biolegend), Rat IgG2a-PerCPCy5.5 (Biolegend) and mIgG1-AF488 (Biolegend) antibodies were used as isotype-matched negative controls.
Zombie NiR (Biolegend) was used to exclude cell death. Samples were acquired on a BD Canto II (BD Biosciences) and CytoFlex S systems (Beckman Coulter). Analyses were performed using FlowJo (Tree Star) and CytExpert software (Beckman Coulter).