Fitc labeled anti cd14

TAMs from malignant ascites or pMPHs from peritoneal lavage fluid were stained with FITC-labeled anti-CD14 (Miltenyi Biotech), APC-labeled anti-CD206 (BioLegend), APC-labeled anti-HLA-DR or APC-labeled anti-CD206 (Biozol), PE-labeled anti-CD163, PE-labeled anti-CD64, PE-Cy7-labeled anti-CD16 and APC-labeled anti-CD32 (eBioscience) as described previously [4 (link)]. Intracellular staining was performed with PE-labeled anti-IL-10 (BD Biosciences) after permeabilization for 20 min at 4 C using BD Cytofix Cytoperm Plus Fixation Permeabilization Kit (BD Biosciences). Additionally, APC-labeled anti-CD52 or APC-labeled anti-TIMD4 (Biolegend) was used for surface staining of TAMs and MDMs from healthy donors. Isotype control antibodies were from BD Biosciences, Miltenyi Biotech and eBioscience. Cells were analyzed by flow cytometry and results were calculated as percentage of positive cells and mean fluorescence intensities (MFI).

Phenotypic analysis was carried out by immunofluorescence using standard procedures. Mouse monoclonal antibodies used for cell-surface staining included FITC-labeled anti-CD14 (Miltenyi), anti-CD115 (R&D Systems) and FITC-labeled anti-CD163 (MBL International Corp., MA). Alexa Fluor-647-labeled isotype-matched secondary antibody (Jackson ImmunoResearch) was used to stain anti-CD115. Control γ₁ FITC/γ_2a PE (BD) was included as a negative control.

Freshly isolated PBMCs and those treated with IronQ were collected in PBS and subsequently blocked for 30 min at 4 °C by using a blocking reagent (PBS containing 2.5% BSA). Cell suspensions were subjected to staining in PBS containing 0.1% BSA at a concentration of 5 × 10⁵ cells. Each 100 µL sample of the cell suspension was incubated with the appropriate IgG isotype controls for each fluorescence channel or with specific antibodies, including FITC-labeled anti-CD34 (Elabscience), FITC-labeled anti-CD14 (Miltenyi), FITC-labeled anti-CD11b (eBioscience), PE/Cy5.5‐labeled anti-CD45 (eBioscience), FITC-labeled anti-CD31 (Life Technologies), PE-labeled anti-CD105 (eBioscience), PE/Cy5.5‐labeled anti‐CD3(clone: UCHT1, Merck Millipore), APC‐labeled anti‐CD206 (clone 15–2, Sigma-Aldrich), APC‐labeled anti‐CCR2 (CD192, Sino Biological), PE‐labeled anti‐CD8a (clone: RPA‐T8, Merck Millipore), PE‐labeled anti‐CD4 (clone: RPA‐T4, Merck Millipore), and Alexa Fluor‐488‐labeled anti‐CD25 (Clone: BC96, eBioscience). This incubation was performed at 4 °C in the dark for 30 min. After two washes containing 0.1% BSA with PBS, the cell suspension was subjected to analysis by using flow cytometry (Beckman Coulter, Epics XL-MCL, CA, USA). Data analysis was carried out by using the FlowJo10 software (Tree Star, Ashland, Oregon), and scattergrams were gated based on three different cell sizes.