Anti cd80 pe l307.4

The expression of HLA class I, costimulatory molecules, and CMV pp65-GFP on aAPCs and aAPC-pp65 was analyzed by flow cytometry. The following antibodies were used in this study: anti-HLA class I (APC; G46-2.6), anti-CD83 (PE; HB15e), and anti-CD80 (PE; L307.4) purchased from BD Biosciences and anti-4-1BBL (PE; 5F4) purchased from BioLegend. Approximately more than 10,000 cells were acquired and the data were analyzed using FLOWJO software (Tree Star, USA).

cDC2s isolated from buffy coats were plated at a density of 0.5 × 10⁶/mL in a 96-well round-bottom plate. Cells were left unstimulated or were treated with MSK1 inhibitors [H89, 10 μM (Bio-Techne); SB 747651A, 10 μM (Bio-Techne); or Ro 31-8220, 5 μM (Sigma)] for 1 h. Then, cells were stimulated with TLR4L at a final concentration of 100 ng/ml. After 6 h, supernatants were stored and cells were lysed for RNA extraction or processed for flow cytometry.
After harvesting, cells were washed in Annexin V Binding Buffer and stained with Annexin V–APC, 7-AAD–PerCP (all from BD Biosciences), anti-CD80–PE (L307.4, BD Biosciences), anti-CD83–FITC (HB15a, Beckman Coulter) and anti-CD86–PB (IT2.2, Sony Biotechnology). Data acquisition was performed using a FACSCanto II flow cytometer (BD Bioscience) and data were analyzed using FlowJo software (Tree Star). The percentage of viable cells after stimulation was measured as the proportion of Annexin V/7AAD double negative cells (Supplementary Figure 3). The expression of co-stimulatory molecules given by the mean fluorescent intensity was evaluated within the viable cells.