Alexa fluor 700 anti mhcii

Twenty-four hours after culture with GM-CSF and/or N-α-Syn stimulation, BMDCs were detached by scraping, washed, and resuspended in 10 μg/ml rat gamma globulin in flow staining buffer (FSB) (0.5% bovine serum albumin (BSA) and 0.1% sodium azide in DPBS) for 40–60 min on ice to block Fc receptors. BMDCs were stained with the following mixture of AlexaFluor 488-anti-CD11c, PECy7-anti-CD11b, PE-anti-Jagged-1, APC-anti-OX40L, AlexaFluor 700-anti-MHC II, eFluor 450-anti-CD86, eFluor 710-anti-CD39 (eBioscience, San Diego, CA) and APC-Vio 770-anti-CD73 (Miltenyi Biotec, Auburn, CA) for 30 min at 4 °C. Cells were washed two times in FSB and were fixed with 1% formaldehyde in DPBS. Samples were analyzed with a BD LSR II flow cytometer and FACSDiva software (BD biosciences, San Jose, CA) at the University of Nebraska Medical Center Flow Cytometry Research Facility. From the single cell-gated population, the percentages of positive for CD11c and CD11b were determined by drawing a gate that comprised 98% of the isotype control as negative. The geometric mean fluorescent intensity (MFI) of each surface marker was determined for CD11c+ cells.

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.