Pe cy7 conjugated cd11c

To analyze M1Mϕ and M2Mϕ polarization, BMDMs were probed with antibodies against phycoerythrin (PE)-conjugated F4/80 (565410; BD Pharmingen, San Diego, CA, USA), FITC-conjugated CD11b (557396; BD Pharmingen), PE-Cy7-conjugated CD11c (558079; BD Pharmingen), and Alexa Fluor-647-conjugated CD206 (565250; BD Pharmingen) at room temperature. TECs were stained using the propidium iodide necrosis detection kit (eBioscience, San Diego, CA, USA). All stained cells were analyzed by flow cytometry (BD Biosciences) using FlowJo software (v.10.0; Tree Star, Ashland, OR, USA).

9 ml of peripheral venous blood was collected from each woman in lithium heparin-treated tubes (BD Vacutainer, BD Biosciences, San Jose, CA). Peripheral blood mononuclear cells (PBMCs) were isolated by standard density gradient centrifugation (Ficoll Paque, Amersham Biosciences AB, Uppsala, Sweden; 25 min, 400 g, room temperature). Cells were kept at −80°C in Fetal Bovine Serum containing 10% DMSO until analysis. After thawing, cells were washed twice in phosphate-buffered saline.
PBMCs were incubated for 30 min at room temperature in the dark with the following antibodies: APC-Cy7-conjugated CD4, PerCP-conjugated HLA-DR, APC-conjugated CD62E, PE-Cy7-conjugated CD62L, PE-conjugated CD62P, FITC-conjugated CD25, PE-Cy7-conjugated CD11c, PE-conjugated CD122, and APC-conjugated CD69 (BD Biosciences). After washing, fluorescent data were registered on a BD FACSAria flow cytometer (BD Biosciences). Data were processed using the FACSDiVa software. 200000 cells per sample were recorded.

DCs were seeded in 6-well plates at 1 × 10⁶ per well and incubated for 24 h with LBP (0, 50, 100, and 200 μg/mL) and LPS (100 ng/mL). DCs were collected and stained with PE-Cy7-conjugated CD11c, V500-conjugated MHCII, BV421-conjugated CD86, and Percp-Cy™ 5.5-conjugated CD80 (BD Bioscience, USA). Stained cells were analyzed by FCM and displayed as mean fluorescence intensity (MFI), and the higher MFI means higher expression of detected molecules.