Tlr2 pe

Flow cytometry was used to find out the percentage of monocytes and DCs that express TLR2. The process used for sampling was as follows: for each participant, a whole blood sample of 100 μL and fluorochrome-conjugated monoclonal antibodies underwent incubation in the dark for 20 min against these antigens:□

CD1c (BDCA-1) FITC/Pacific Blue anti-Human Lineage Cocktail (anti-CD3, CD14, CD16, CD19, CD20, CD56)/TLR2 PE (Biolegend, San Diego, CA, USA);

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BDCA-2 FITC/CD123 Pe-Cy7/CD45 V450/TLR2 PE (Biolegend);

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CD14 FITC/CD16 V450/HLA-DR Pe-Cy7 (BD Biosciences, San Jose, CA, USA) and TLR2 PE (Biolegend).

Thereafter, Lysing Buffer (BD Pharm Lyse) was used to treat the samples, and they were then washed in PBS solution (Sigma-Aldrich, St. Louis, MO, USA). The Cytoflex LX (Beckman Coulter, Brea, CA, USA) was used to collect the samples. The data were analyzed using the Kaluza Analysis software and evaluated with dot plots. The definition of mDCs was BDCA1+ Lin− cells; plasmacytoid DCs, BDCA2+ CD123+ cells; classical monocytes, CD14+ CD16− cells; and non-classical monocytes, CD14+ CD16+. A gating step with HLA-DR was added to improve the monocyte purity and isotype controls (Biolegend) used for the determination of binding that was unspecific.

Single cell suspensions obtained from full thickness samples or after stimulation with S. epidermidis and MRSA were first labeled with live/dead detection kit (Yellow Amine, Thermo Fisher Scientific) and then with the following fluorescently labeled antibodies: CD45-Alexa Fluor 700, TCR GD-PE-Cy7, CD31-PacBlue, CD104-FITC, CD325-PerCPCy5.5, and CCRL1-PE (Biolegend, San Diego, CA, United States). We also stained cells with fluorescently labeled antibodies for TLR1-BV570, TLR2-PE, TLR6-BV605, and TCR GD 1 FITC (Biolegend, San Diego, CA, United States). P-2 mRNA was detected using an amplified signal FISH technique (PrimeFlow; Affymetrix/eBioscience-Thermo Fisher Scientific). For mRNA detection, target probe hybridization was performed using type 1 (AlexaFluor647) probes for P-2 as described (43 (link)). Approximately 20,000 cell events were acquired from each sample on flow cytometer equipped with 405 nm, 488 nm, 642 nm, and 785 nm (SSC) lasers (Fortessa X-50, BD Immunocytometry Systems, San Jose, CA, United States). Spectral compensation was completed using single color control samples and antibody capture beads (BD Biosciences). Data were analyzed using FlowJo version 10.2 (TreeStar).

Cell surface expression of scavenger receptors CD36, CD163, and CD206, and cell surface expression of TLR1, TLR2 and TLR4 on monocytes were assessed by staining PBMCs with CD14-PerCP/Cy5.5, CD36-PE, CD163-APC, CD206-Alexa Fluor 488, TLR2-PE, TLR4-APC (BioLegend), and TLR1-FITC (Invivogen) fluorochrome-conjugated antibodies according to the manufacturers’ instructions. Samples were fixed in 2% paraformaldehyde solution and were run on the flow cytometer. Percent expression of scavenger receptors and MFI of TLRs were measured after gating on CD14⁺ monocytes.