Apc anti human cd14 antibody

Before the induction of apoptosis, Jurkat T-cells were stained with 5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE, Life Technologies, Milan, Italy) for 30 minutes at 37 °C. CSFE-labeled apoptotic cells (1 × 10⁶ cells/ml) were co-cultured with MDMs (2:1 ratio) for 30 minutes. Non-ingested cells were removed. Adherent MDMs were detached with trypsin, incubated with APC anti-human CD14 antibody (BD Biosciences), or isotype-matched irrelevant antibody, for 15 minutes at room temperature (RT), and analyzed by flow cytometry. 10,000 events were acquired, and the data were analyzed using CellQuest software (Becton-Dickinson). Data are expressed as percentage of CD14-positive MDMs that had binding/engulfed CSFE-labeled apoptotic Jurkat T-cells^{54 (link)}.

Monocytes were washed in 1× PBS and incubated in blocking solution consisting of FACS buffer (145 mM NaCl, 8.45 mM Na₂HPO₄, 1.83 mM NaH₂PO₄, and 0.1% NaN₃), 5% BSA, and human FcR binding inhibitor (eBioscience, San Diego, CA, USA) for 20 min on ice. After blocking, cells were stained by adding APC anti-human CD14, Alexa Fluor 488 anti-human CD16, PE/Cy7 anti-human CD163, Brilliant Violet 605 anti-human CD86 (BioLegend, San Diego, CA, USA), and APC-R700 anti-human CD80 antibodies (BD Biosciences, San Jose, CA, USA) or isotype controls on ice. Cells were then washed in 1× PBS and analyzed by flow cytometry using an LSRFortessa cell analyzer and BD FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA).
For viability studies, monocytes were washed after incubation with the APC anti-human CD14 antibody as described above. Cells were then stained with phycoerythrin-annexin V (BD Pharmingen, Franklin Lakes, NJ, USA) and either Sytox Blue dead cell stain (Life Technologies, Carlsbad, CA, USA) or propidium iodide (PI) dead cell stain (Thermo Fisher Scientific, Waltham, MA, USA) to detect dead and dying cells. After staining, cells were analyzed by flow cytometry as described above. Double negative cells represent live cells, whereas double positive and single positive cells represent dead and/or dying cells.