Cd163 pe

Cells cultured in the absence and presence of human M-CSF were processed at minimum five days after plating for flow cytometry. Cells that remained in suspension were collected whereas adherent cells were detached with StemPro Accutase (ThermoFisher). For both conditions, the cell suspension was centrifuged and the supernatant removed as previously described. The cell pellets were re-suspended in ice-cold PBS with the following antibodies to assess surface expression for determination of macrophage polarization. Cells were incubated on ice with CD14-FITC (BD Biosciences, San Jose, CA), CD86-PE (BD Biosciences), and CD206-APC (BD Biosciences), or with CD14-FITC, CD163-PE, and CD206-APC antibodies for 1 hour and cells were washed twice before processing for flow cytometry. Unstained cells and cells stained with single antibodies were used for gating cell size and setting instrument parameters. Samples were processed on a BD FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). Unstained cells were used to indicate background fluorescence and to set quadrants before calculating the percentage of positive cells.

After the polarisation, THP-1-derived macrophages were harvested and washed in PBS. 1 × 10⁶ cells were then stained using anti-CD11b-PE-Cy7, CD209-FITC, CD163-PE, CD115-PE-Cy7, CD204-FITC, CD206-APC (BD Pharmingen, USA) for 30 min at 4 °C. 1 × 10⁶ cells isolated from fresh xenograft tissues were stained using FITC-anti-mouse F4/80 (Bio-Rad), FITC-anti-mouse CD206 (Biolegend). An isotype-matched IgG was used as negative control. Results were analysed by flow cytometry (FACSCalibur, BD).

For the isolation of human monocyte-derived macrophages (hMDMs), venous blood was collected from healthy human volunteers and the mononuclear fraction isolated after density gradient centrifugation (400g for 20 min) using Ficoll Paque-plus (GE healthcare). Cells were plated at a density of 2.5×10⁶/ml in RPMI-1640 (Gibco) without FBS for 2 hours. Non-adherent cells were discarded after 2h and replaced with RPMI-1640 supplemented with 10% (v/v) FBS incubated for 7d, with media changed every 3d. To obtain M2 polarized cells, 40 ng/ml IL-4 (Peprotech) was added after the initial culture for 72h [17 (link)]. Macrophages were then detached from the plates using 2.0 mg/ml Dispase II (Sigma) [15 (link)]. For differentiation analysis, cells were incubated with CD14-FITC (BD Pharmingen) and CD163-PE (BD Pharmingen). Cell samples were then run on a BD FACSCalibur (BD, USA).

Blister and circulating leukocytes were enumerated and then analysed for surface marker expression on a flow cytometer (LSR Fortessa, BD Biosciences). Due to a lack of published data on blister leukocyte differentiation using flow cytometry, a novel staining and subsequent gating strategy was designed to identify individual cell populations. Leukocytes were incubated with combinations of antibodies to CD3 (APC, Clone: UCHT1, BD), CD19 (PE-Cy 7, Clone: SJ25C1, BD), CD56 (PerCP-Cy5.5, Clone: B159, BD), HLA-DR (V450, Clone: L243, BD), CD14 (Alexa Fluor 700, Clone: M5E2, BD), CD16 (FITC, Clone: 3G8, BD), CD141 (PE, Clone: M80, Biolegend), CD163 (PE, Clone: M130, BD), CD11c (PE-Cy7, Clone: B-ly6, BD), Siglec-8 (PE, Clone:7C9, Biolegend) and Annexin-V/7AAD Apoptosis Detection Kit (BD) using respective isotype antibodies and fluorescence-minus-one (FMO) controls, and compensated for dual labelling. Separation of cell subtypes was performed using a cell sorter (FACS Aria, BD Biosciences) with subtypes undergoing histological staining using a modified Wright's method (Shandon Kwif-Diff Stain Kit, Thermo Scientific). Flow cytometry analysis was completed using FlowJo software (Tree Star Inc).

The expression of surface markers (CDs) in macrophages was analyzed using flow cytometry. After differentiation, macrophages were stimulated with LPS and exposed to treatments for 4 days. After that, cells were harvested with Stem Pro™ Accutase™ cell dissociation reagent (ThermoFisher Scientific, Waltham, MA, USA), collected by centrifugation in the cold, and washed once with FACS buffer prepared with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at pH 7.4, 140 mM sodium chloride (NaCl), and 2.5 mM calcium chloride (CaCl₂). Cells were incubated with fluorochrome-conjugated antibodies (1:50 dilutions) in 50 μL of FACS buffer for 15 min in the dark. Cells were stained separately in each single screening tube with a cluster of differentiation (CD)80-PE and CD163-PE (all purchased by BD Biosciences, MA, USA) Then, the excess antibodies were removed by adding fresh FACS buffer and centrifugation. After that, 20,000 events were run in a Beckman Coulter CytoFLEX flow cytometer (Brea, CA, USA). Relative fluorescence emissions of gated cells by forward and side scatter properties (FSC/SSC) were analyzed using the CytExpert Software (Beckman Coulter) and expressed as the MFI ratio on the isotype control. Individual values obtained from three independent experiments (n = 3) were summarized as means and standard deviations.