Cd11c pe cy5

Twenty-four hours after LPS stimulation, BMDCs were detached from the plate with D-PBS 1X (Gibco, New York, NY, USA) + 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA), washed with DPBS 1X + 0.5%BSA (Sigma-Aldrich, St Louis, MO, USA), and stained with CD11c PE Cy5 (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD86 PE (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Flow cytometer acquisition was performed using the NAVIOS flow cytometer (Beckman Coulter, Brea, CA, USA), and data analysis was performed using Kaluza software, version 1.5a (Beckman Coulter, Brea, CA, USA). Gating strategy: BMDCs were gated based on the physical properties and checked for positivity to CD11c (forward scatter vs. CD11c). BMDCs CD11c⁺ gated cells were analyzed for CD86 expression staining by histogram.

Next, 24 h after LPS stimulation, BMDCs were detached from the plates with DPBS 1× (Gibco, MA, USA) + 0.5mM EDTA (Thermo Fisher Scientific, MA, USA). Cells were then washed with DPBS 1× + 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) and labelled with CD11b VioBrightFITC (Miltenyi Biotec, Bergisch Gladbach, Germany), MHCII PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD11c PECy5 (Miltenyi Biotec, Bergisch Gladbach, Germany), F4/80 APC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD80 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD86 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), and 7-AAD PECy5 (Miltenyi Biotec, Bergisch Gladbach, Germany). Flow Cytometer data analysis was performed using NAVIOS software (Beckman Coulter, Brea, CA, USA), with at least three experiments performed. Flow cytometer analysis was performed using Kaluza Software 1.5 (Beckman Coulter, Brea, CA, USA).