Cd1a alexa fluor 488

Fluorescence-conjugated antibodies were used to stain DCs: ACE2-APC, CD1a-Alexa Fluor 488, CD40-APC, CD80-PerCP/Cy5.5, CD83-PE, CD86-Alexa Fluor 488, C-X-C chemokine receptor type 4 (CxCR4)-PE, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)/CD209-PerCP/Cy5.5, human leukocyte antigen (HLA)-DR-PE, HLA-ABC-APC, and programmed death-ligand 1 (PD-L1)-FITC (all from BioLegend). Isotype-matched antibodies were used as controls. Shortly, DCs were washed and resuspended in phosphate-buffered saline (PBS) + 1% FBS. Then, 3 µL of fluorescence-conjugated antibodies were added to cells and incubated for 30 min, at 4 °C, in the dark. Cells were subsequently washed, resuspended in PBS + 1% FBS, and analysed in an Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA). Data were analysed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA), and the results are presented as mean fluorescence intensity (MFI), obtained by the subtraction of isotype control values.

Cell staining was performed using fluorescence-conjugated antibodies, specifically CD1a-Alexa Fluor 488, CD14-PE, CD11c-APC, CD1c-FITC, CD16-APC, CD86-Alexa Fluor 488, CD83-PE, CD80-PerCP/Cy5.5, CD40-APC, human leucocyte antigen (HLA)-DR-Alexa Fluor 488, HLA-ABC-APC, CCR C-C chemokine receptor 1 (CCR1)-Alexa Fluor 488, CCR2-PerCP/Cy5.5, CCR5-APC, CCR7-PerCP/Cy5.5, chemokine receptor (CXCR4)-PE, CD3-PE, CD4-PerCP/Cy5.5, CD8-APC, CD69-FITC, CD25-APC, forkhead-box-P3 (FoxP3)-FITC, T-box protein expressed in T-cells (T-bet)-PE (all from Biolegend). Isotype-matched antibodies were used as controls. Briefly, 0.2 × 10⁶ monocytes, DCs or T cells were washed and stained with 3 µl of fluorescence-conjugated antibodies in phosphate-buffered saline (PBS) + 1% FBS for 30 min at 4°C, in the dark. Cells were subsequently washed, resuspended in PBS + 1% FBS and analyzed in an Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA). For intracellular staining, Fix&Perm (Thermo FisherScientific, Waltham, MA, USA), a fixation and cell permeabilization kit, was used as described by the manufacturer. Data were analyzed with FlowJo™ software (version 10) and results presented as percentage of positive cells or mean fluorescence intensity (MFI) after subtraction of isotype control values.