Apc anti human cd14

Cell surface staining of blood mononuclear cells and isolated monocytes was carried out by incubation with FITC antihuman CD14 and APC anti human CD14 (BioLegend, San Diego, CA, US). Expression of protein levels of IRAK-M was determined by flow cytometry intracellular staining. THP-1 cells and PBMCs were fixed and permeabilized (Transcription Factor Staining Buffer Set, Thermo Fisher Scientific, Waltham, MA, US). Then, staining was carried out with anti-human IRAK-M rabbit polyclonal antibody (ab-8116, Abcam) and anti-rabbit APC conjugated secondary antibody (BioLegend, San Diego, CA, US). The proper isotype control was used as a control. The flow cytometry events were acquired in a FACS Calibur (BD Biosciences, San Jose, CA) and analyzed with the use of Summit v4.3 Software.

For flow cytometry analysis of CD45⁺ depletion in the ovarian cancer ascites sample, cells were resuspended in PBS complemented with 2% FBS and stained with FITC anti-human CD45 antibody (BioLegend, cat. no. 304006CD45; 1:200 dilution), PE anti-human EpCAM antibody (Miltenyi Biotech, cat. no. 130-113-264; 1:50 dilution), APC anti-human CD14 (BioLegend, cat. no. 367118, clone 63D3; 1:20 dilution) and PE-cy7 anti-human CD24 (BioLegend, cat. no. 311120, clone ML5; 1:20 dilution) for 20 min, and with 7-AAD (Invitrogen, cat. no. A1310; 1:200 dilution) for 5 min. The same cells were also used for single-stain and unstained controls to perform compensation and adjust gating. Analysis was performed on a BD LSRFortessa cell analyzer with BD FACSDiva Software Version 8.0.1 and plots were generated with FlowJo Version 10.5.3. Gating for CD45 and EpCAM was performed as described in Extended Data Fig. 4c. CD24 and CD14 antibodies were included in the antibody panel for FACS analysis to provide additional information and better inform scRNA-Seq. Specifically, expression of CD24 on tumor cells has been shown to relate to ovarian cancer invasiveness and expression of CD14 identifies monocytes/macrophages.

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.