Cd4 af488

Immature and mature LCs were phenotyped using CD1a-APC (BD Pharmingen), langerin-PE (Novocastra), CD86-FITC (BD Pharmingen), CD80-PE (BD Pharmingen), CD83-APC (BD Pharmingen), CD4-AF488 (Biolegend), CD195 (CCR5)-PE (BD Pharmingen) and unlabeled CXCR4 (R&D systems) for which secondary detection with Goat-anti-Mouse-A488 (Invitrogen) was used. HIV-1 infection and transmission samples were stained for CD1a-APC (LC marker), CD3-PerCP (T cell marker), and p24-PE (HIV-1 envelope protein, Beckman Coulter). Immature vaginal LCs were further sorted with a FacsARIA 3 laser sorter (BD Biosciences) after staining for CD1a-APC into CD1a positive and CD1a negative fractions. Samples were analysed using FACSCanto II flow cytometers (BD Biosciences) and data analysis was carried out with FlowJo V10.

Single-cell suspensions were obtained from individual mice and cell phenotypes were analyzed by flow cytometry as described previously (25 (link)) using the FACS Calibur machines (BD Biosciences). The gating strategy is displayed in Figure S1. The following labeled monoclonal antibodies were used: CD4-PErCp (dilution 1:300), CD4-AF488 (dilution 1:200), or CD4-PE (dilution 1:200) (all GK1.5, BioLegend), CXCR5-Fitc (L138D7, BioLegend, dilution 1:100), CD19-AF647 (6D5, BioLegend, dilution 1:400), B220-APC (RA3-6B2, BioLegend, dilution 1:400), F4/80-AF-488 (BM8, BioLegend, dilution 1:200), Ly6G-PE (1A8, BD Biosciences, dilution 1:200), CD44-FITC (IM7, BioLegend, dilution 1:150), CD62L-PE (MEL-14, BioLegend, dilution 1:200), CD11b-biotine (M1/70, dilution 1:100) with subsequent SAv-PerCP (BioLegend, dilution 1:500) staining. For intracellular staining of the lung cells, BD Fixation/Permeablization Kit (BD Biosciences) was used due to the recommendations. Briefly, after 16-17 hours of in vitro cultivation in the presence of mycobacterial antigens, cells were harvested, stained first for surface antigens, then fixed with Cytofix/cytoperm buffer for 30 minutes and then stained for intracellular cytokines with IFN-γ-PE (BD Biosciences dilution 1:150) and IL-17-PerCp (TC11-18H10.1, BioLegend, dilution 1:150) antibodies. Results are presented as mean ± SD.

Cytofluorimetric analysis was performed as previously described [29 (link),30 (link)]. Briefly, hearts were collected and cut into small pieces with a blade, and then incubated with collagenase type IV for 1 h and 30′ at 37 °C with agitation. The obtained cell suspension was passed through a 70 μm and then a 40 μm cell strainer; the cells were then counted on a hemacytometer, collected by centrifugation at 1200 rpm, and suspended in 200 μL of calcium/magnesium-free PBS (phosphate-buffered saline) with 2% FBS (foetal bovine serum). They were then divided into two tubes for the staining. The cells were then incubated on ice for 30 min with the following antibodies: CD45 PE/Cy7, F4/80 APC, Ly6g PE Fluor 610, CD11b APC/Cy7, CD206 PERCP/Cy5.5 Ly6c BV-510, I-Ab FITC (tube 1) and CD45 PE/Cy7, CD3 PERCP Cy5.5, B220 BV-510, CD4 AF488, and CD8 PE (tube 2), all by Biolegend, (San Diego, CA, USA). Cells were then washed with 3 mL of calcium/magnesium-free PBS and resuspended in 200 μL of calcium/magnesium-free PBS with 2% FBS. Samples were acquired with a CyAn ADP (Agilent DAKO, Santa Clara, CA, USA), and acquired data were analysed using FlowJo software version 10.1.