Anti il17 percp cy5

Approximately 75 × 10³ cells were transfected (n = 6) with miRNA 548a-3p mimic or inhibitor or negative control in a U-bottom 96-well plate and incubated for 24 h in cell culture medium containing 10% heat inactivated FBS. This was followed by replacing the medium with fresh culture medium and induction with anti-CD3/28 (StemCell, Vancouver, Canada) for additional 24 h. After the incubation was over, cells were stained with anti-CD4 APC/Fire 750, anti-CD25 Pacific blue and anti-FoxP3 FITC, anti IL17 Percp/Cy5.5, anti IFNγ PE/Cy7 (BioLegend, San Diego, CA, USA), anti IL10 BV711 (BD Biosciences, San Jose, CA, USA), anti Akt2 AF647 (R&D System, NE Minneapolis, MN, USA) and fix viability dye (Invitrogen, Waltham, MA, USA) as previously described [14 (link)]. After staining was done, cells were resuspended in FACS buffer. Cell samples were then run in LSRFortessa™ Cell Analyzer (BD Biosciences) and data were analyzed using FlowJo software.

To assess in vivo macrophage numbers, digested cells and splenocytes were stained with fluorescence conjugated antibodies against MHC II, CD45, F4/80, and CD11b (eBioscience, San Diego, CA). LIVE/DEAD staining (Thermo Fisher Scientific) was used to assay for cell viability determination at 405 nm excitation. For intracellular cytokine staining, freshly isolated IELs and splenocytes were incubated with 50 ng/ml PMA (Sigma), 750 ng/ml ionomycin (Sigma, St. Louis, MO) and 5 μg/ml Brefeldin A (Biolegend, San Diego, CA) at 37°C for 4 h. Cells were first collected for surface staining with anti-CD8-BV650 (Biolegend, San Diego, CA), anti-CD62L-BV605 (Biolegend, San Diego, CA), anti-CD4-PE Cy7, and anti-CD44-APC (Thermo Fisher Scientific, Waltham, MA) antibodies, and then fixation and permeabilization were performed following the instructions from BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences, San Jose, CA). Intracellular staining for cytokines was detected with anti-IFN-γ-PE, anti-TNF-α-FITC, and anti-IL-17-Percp Cy5.5 antibodies (Biolegend, San Diego, CA). Data were collected using a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (FlowJo, LLC, Ashland, OR).