Apc cy7 anti cd3

Single-cell suspension was prepared from the spleen in PBS for flow cytometry analyses. Cells were counted using an automated cell counter (Sysmex Europe GmBH, Sweden). Flow cytometry analysis was performed using different fluorochrome-conjugated antibodies i.e. APC-anti-CD267 (TACI), PerCP-anti-CD19, BV421-anti-CD138, V500-anti-B220, and APC-Cy7-anti-CD3, all purchased from eBioscience (Sweden). Analysis was performed using the BD FACS-verse flow cytometer and Flow Jo software (FlowJo10.6.2).

For intracellular cytokine detection, isolated cells were cultured in 20 μg/ml of M. tuberculosis lysate or PMA/ionomycin for 1.5 h before 10 μg/ml Brefeldin A (eBioscience, USA) was added to the culture for 3.5 h more. For surface staining, lymphocytes were harvested, washed and stained for 30 min on ice with mixtures of fluorescently conjugated mAbs or isotype-matched controls. mAbs of mice were as follows: APC-Cy7-anti-CD3, PE-Cy7-anti-CD4, APC-anti-CD8a, PE-anti-CD45.1, Alexa Fluor 700-anti-CD45.2, PE-anti-CD25, PerCP-cy5.5-anti-CD69, PE-anti-CD44, FITC-anti-CD62L, PE-cy7-anti-Gr-1, PE-anti-CD11b, APC-ant-CD86, Alexa Fluor 700-anti-MHC-II and PerCP-cy5.5-anti-CD206 (eBioscience). For intracellular staining, the cells were incubated 20 min in IC Fixation buffer (eBioscience), followed by permeabilization buffer (eBioscience) and 1 h of incubation with appropriate mAbs of mice: PE-anti-IFN-γ, FITC-anti-IL-17, PerCP-cy5.5-anti-IL-4, FITC-anti-IL-2 and FITC-anti-Foxp3. Cell phenotype was analyzed by flow cytometry on a flow cytometer (BD LSR II) (BD Biosciences, USA). Data were acquired as the fraction of labeled cells within a live-cell gate and analyzed using FlowJo software (Tree Star). All gates were set on the basis of isotype-matched control antibodies.