Fluorescein isothiocyanate conjugated anti cd4

The following fluorochrome-conjugated anti-mouse monoclonal antibodies for cell surface markers and intranuclear factor were purchased from eBiosciences (San Diego, California, USA): fluorescein isothiocyanate-conjugated anti-CD4 (cat no. 11-0041-85), anti-Gr-1 (cat no. 11-5931-82), anti-MHC-II (cat no. 11-5321-82); phycoerythrin-conjugated anti-CD45 (cat no. 12-0451-83), anti-CD25 (cat no. 12-0251-83); phycoerythrin cyanine7-conjugated anti-CD8 (cat no. 25-0081-82), anti-F4/80 (cat no. 25-4801-82); allophycocyanin (APC)-conjugated anti-CD11b (cat no. 17-0112-82), and anti-Foxp3 (cat no. 17-5773-82). Single-cell suspensions of splenocytes, tumor-infiltrating lymphocytes, and ascitic cells were stained on ice for 30 min with the indicated cell surface marker antibodies (dilution, 1: 200). For intranuclear Foxp3 staining, cells were fixed and permeabilized using a Cytofix/Cytoperm Kit (cat no. 00-5523-00; eBiosciences) on ice for 30 min after labeling with surface marker antibodies, followed by anti-Foxp3 mAb (dilution, 1: 50) intranuclear staining on ice for 30 min. Samples were acquired on a BD FACScanto II flow cytometer (BD Biosciences, San Jose, California, USA) and the results were analyzed using Flowjo software (TreeStar, Ashland, Oregon, USA).

After 5 days of culture, splenocytes were stained with fluorescein isothiocyanate-conjugated anti-CD4 (eBioscience, Fisher Scientific, Loughborough, UK) and Pacific blue-conjugated anti-CD25 (Biolegend, London, UK) antibodies, permeabilized and stained with phycoerythrin-conjugated anti-FoxP3 (eBioscience). Samples were analyzed on an LSR Fortessa™ flow cytometer (BD Bioscience, Oxford, UK) using FACSDiva™ software. Regulatory T cells were defined as CD4⁺CD25⁺FoxP3⁺.^{40 (link)}

A single-cell suspension was prepared from the cervical lymph nodes removed from three mice. After lysis of red blood cells in 1 × RBC Lysis Buffer Solution (eBioscience, San Diego, CA, USA) and selection of live cells using the Zombie Yellow Fixable Viability Kit (BioLegend, San Diego, CA, USA), lymphocytes were incubated with mouse anti- CD16/32 antibody (Tonbo Biosciences, San Diego, CA, USA) followed by incubation with fluorescein allophycocyanin-conjugated mouse anti-CD8a (BD Biosciences, San Jose, CA, USA), phycoerythrin-Cy7-conjugated mouse anti-CD69 (BioLegend), BrilliantViolet785-conjugated anti-mouse CD3 (BioLegend), fluorescein isothiocyanate-conjugated anti-CD4 (eBioscience), or fluorophore-conjugated isotype controls (eBioscience). The cells were analyzed using CytoFLEX (V5-B5-R3 configuration, Beckman Coulter, Fullerton, CA, USA), and data were analyzed with CytExpert software (Beckman Coulter).