Cyto fast fix perm kit

For flow cytometry analysis, 2x10⁵ isolated cells per sample were incubated in 100 µl MACS buffer for 20 minutes in the dark with different sets of antibodies, as specified in Supplementary Table 1. For intracellular labelling, cells were fixed and permeabilized using the Cyto-Fast™ Fix/Perm kit from BioLegend^® San Diego, CA, USA, according to the manufacturer’s protocol prior to incubation with antibodies. 4′, 6-diamidino-2-phenylindole (DAPI) was added just before the analysis for live/dead cell discrimination. All flow cytometry investigations were performed on a BD LSRFortessa™ X-20 device (BD Biosciences, Heidelberg, Germany) with FACS Diva software, and the data sets were analyzed using the FlowJo^® V10 software (BD, Ashland, OR USA).

RAW264.7 cells were cultured at high and low density (2500 and 400 cells/mm² respectively) overnight in a 6-well plate (CellTreat 229106) in DMEM media supplemented with 10% FBS. Cells at each density were divided into five aliquots of equal cell count (approximately 0.5 x 106) and washed with PBS, stained with a live dead reactive dye (BioLegend L34975), fixed and permeabilized with Cyto-Fast Fix/Perm Kit (BioLegend 426803) following kit instructions, and then blocked with anti-mouse CD16/32 antibody (BioLegend 101319) at 10 ug/mL. For each density, three samples were stained with PE anti-mouse CD287 (TLR7) Antibody (BioLegend 160003) at 5 ug/mL, one with PE Mouse IgG1, κ Isotype Ctrl (FC) Antibody (BioLegend 400113) at 5 ug/mL, and one left unstained, at the steps indicated in the Fix/Perm protocol. The cells were then washed and resuspended in Cell Staining Buffer (BioLegend 420201) and run on an ACEA NovoCyte Flow Cytometer.