Cd44 bv421

Cells isolated from spleen, lungs or peripheral blood were cultured with 2 µg/ml of two immunodominant peptides (Pepscan, Lelystad, The Netherlands), [SSTHEANTMAMMARDT] and [AGYAGTLQSLGAEIAV] of the TB10.4 protein, previously demonstrated to stimulate both CD4⁺ & CD8⁺ T cell responses (Kaveh & Hogarth, unpublished); 1 µg/ml anti-CD28 (BD Biosciences) and 10 µg/ml Brefeldin A (Sigma) for 16 h at 37 °C/5% CO₂. Cells were washed at 300 g for 5 min and surface stained with combinations of CD62L-FITC, CD27-PerCP-Cy5.5, CD8-AF700, CD44-BV421, CD127-PE-Cy7, CD69-FITC, CCR7-BV421, live/dead-Zombie Aqua (all BioLegend) and CD4-APC-H7 (BD Biosciences). Cells were then washed, treated with BD Biosciences Cytofix/Cytoperm as per manufacturer’s instructions and stained intracellularly with combinations of IFN-γ-PE-Cy7, IL-2-APC (both eBioscience), IFN-γ-BV605 and TNF-α-BV605 (both BioLegend). Cells were washed again and analysed using an LSRFortessa™ analyser utilising a 532 nm laser for PE and PE-conjugate excitation with FACSDiva™ software (BD Biosciences). Final analysis was performed using FlowJo® software (Tree Star Inc.) on a minimum of 100,000 live lymphocytes (50,000 for peripheral blood).

Two panels of fluorochrome-conjugated antibodies (BioLegend, San Diego, CA, USA) were used to identify the innate immunity cell populations and to analyze the activation marker expression: (1) CD11b-PE/Cy7, CD11c-PE, MHCII-Alexa488, CD103-PerCP-Cy5.5, CD45-APC/Cy7, CD64-BV421, CD24-BV510; (2) CD45-APC/Cy7, MHCII-Alexa488, Ly6G-PerCP-Cy5.5, CD86-BV421, CD83-BV510. T-cells staining was performed using the fluorochrome-conjugated antibody set containing CD4-PerCP-Cy5.5, CD8-PE/Cy7, CD62L-APC/Cy7, and CD44-BV421 (BioLegend, San Diego, CA, USA). Intracellular production of cytokines was assessed using antibodies against IFNγ-FITC, IL2-PE, and TNFα-BV510 (BioLegend, San Diego, CA, USA). Staining for the detection of intracellular markers was performed using the Fixation and Permeabilization Solution reagent kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Zombie Red viability marker (BioLegend, San Diego, CA, USA) was used to identify the dead cells. True Stain reagent (BioLegend, San Diego, CA, USA), containing antibodies to CD16/CD32, was added during the surface markers staining to block non-specific antibody binding. Data were collected on a Cytoflex flow cytometer (Beckman Coulter, Bray, CA, USA). The results were analyzed using the Kaluza Analysis 2.2 program (Beckman Coulter, Bray, CA, USA).