Cd104 fitc

For FACS analysis, lungs were digested in collagenase (1mg/ml collagenase in 3ml DPBS + 0.2g/L glucose per lung) at 37 °C for 60 min while shaking at 165 rpm, followed by red blood cell lysis with 0.64 % ammonium chloride at 37 °C for 3 min [31 (link)]. Cells were resuspended in blocking solution (anti-FcR and Rat IgG) and incubated on ice for 10 min. Cells were stained with conjugated antibodies CD31-PECy7, CD45-PECy7, EpCAM-APC, CD104-FITC and CD24-PE (Biolegend) as described previously [32 (link)]. Cells were then washed and resuspended in PI solution. Cells were sorted on an ARIA II (Beckton Dickinson).

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).