Anti cd4 fitc clone gk1

The establishment of long-lasting immunity and the subsequent protection against Leishmania parasites is dependent on the production of specific memory and effector T cells. To analyze the lymphocyte population, spleens from vaccinated and infected animals were obtained. Briefly, splenocytes were processed as described below to obtain a single-cell suspension. Subsequently, cells were counted, and 10⁶ cells/mL were incubated in the presence of 25 μg/mL of specific L. infantum or L. major SLAs at 37 °C, 5% CO₂ for 24 h. Cells were then triple-stained with anti-CD4 FITC (Clone GK1.5, Biolegend) or anti-CD8 FITC (Clone 53-6.7, Biolegend) and anti-CD44 (Clone IM7, Biolegend) and anti-CD62L (Clone MEL-14, Biolegend) for 30 min extracellular staining at 4 °C in the dark. Cells were then fixed with paraformaldehyde 4% w/w. Dead cells were excluded using a LIVE/ DEAD Zombie NIR Fixable Viability Kit (Biolegend). Cells were analyzed on a CytoFLEX^® instrument (Beckman Coulter, Life Science, Indianapolis, IN, USA), and the CytExpert™ software package version 2.5 (Beckman Coulter, Life Science, Indianapolis, IN, USA) was used for analysis based on at least 100,000 events per sample.

Upon stimulation and prior to harvest, isolated spleen cells were pre-incubated with brefeldin A (BioLegend, San Diego, CA, USA) for 4 h at 37 °C. To block non-specific antibody binding, the TruStain FcX™ antibody (Clone 96, BioLegend, San Diego, CA, USA) was then added. Subsequently, splenocytes were incubated with Zombie RED (Fixable Viability Kit, BioLegend, San Diego, CA, USA) for 10 min at room temperature, washed and immunostained with anti-CD4 FITC (clone GK1.5, BioLegend) (diluted 1:800) and anti-CD8 PE (clone QA17A07, BioLegend) (diluted 1:800) fluorophore-labeled antibodies. After two washes with PBS containing 2% FCS, cells were fixed and permeabilized using Cyto-FastTM Fix/Perm Solution (BioLegend) according to the manufacturer’s protocol. Then, cells were stained with anti-IFN-γ PerCP (clone XMG1.2, BioLegend) and anti-IL-10 PCy7 (clone JES5-16E3, BioLegend) at room temperature for 30 min, washed twice, and analyzed. The production of intracellular cytokines was expressed as percentages, representing the ratio of the percentage of IFN-γ or IL-10-producing-T cells in SLA-stimulated cultures.

To analyze the proliferation of T cells, CFSE (eBioscience Inc., San Diego, CA, USA) was used. Cell surface MHC class II and CD86 on BMDCs were stained with anti-I-A/I-E-PerCP (clone M5/114.15.2, BioLegend) and anti-CD86-PE (clone GL-1, BioLegend), respectively. Intracellular IFN-γ and IL-4 were stained with anti-IFN-γ-PE/Cyanine7 (clone XMG1.2, BioLegend), and anti-IL-4-PE (clone 11B11, BioLegend), respectively, with anti-CD4-FITC (clone GK1.5, BioLegend) after treatment with the Fixation Buffer (#420801, BioLegend) and the Intracellular Staining Perm Wash Buffer (#421002, BioLegend). Fluorescence was detected by a MACS Quant Analyzer (Miltenyi Biotech) and analyzed with FlowJo (Tomy Digital Biology, Tokyo, Japan).