Anti cd163 pe

For direct immunofluorescence labeling, macrophages were stained with the following fluorescence-labeled anti-human mAbs: anti-CD40-FITC, anti-CD64-A700, anti-CD80-V450 and anti-CD86-APC, anti-CD11b-pacific blue, anti-CD163-PE, anti-CD206-FITC (all from BD Biosciences, San Diego, CA) and anti-CD200R-APC Abs (Serotec, Oxford, UK). For Notch1 receptor analysis, macrophages were immunostained using rabbit anti-Notch1 (1:100 dilution, ab52627 from Abcam, Cambridge, UK) Abs and Alexa-488 labeled anti-rabbit IgG as secondary antibodies. For apoptosis analysis, cells were incubated with Annexin V/propidium iodide following the manufacturer’s recommendations (Life Technologies). Cells stained with, isotype-matched, irrelevant Abs were used as negative controls. Fluorescence was measured by flow cytometry after gating on FSC/SSC parameters using a FACS LSR II® (BD Biosciences) and next analyzed using FlowJo® VX software (Tree Star, Inc., Ashland, OR, USA).

To detect CD11b + CD163+ macrophages, anti-CD163-PE (BD Biosciences, USA) and anti-CD11b-APC (eBioscience, USA) were used to stain cells. Isotype controls were run in parallel. Flow cytometry was performed using a BD Accuri C6 flow cytometer (BD Biosciences).

The following flow cytometry antibodies were purchased from BD Biosciences (San Jose, CA): anti-CD3-PE Cy7, anti-CD4-Alexa488, anti-CD8-PacBlu, anti-CD28-PE, anti-CD57-APC, anti-CD45RO-Alexa 700, anti-HLA-DR-APC, anti-CD14-PacBlu, anti-CD11a-FITC, anti-CD16-PE-Cy7, anti-CD163-PE, anti-CD62L-APC, and anti-CD86-Alexa 700. Two multi-color panels were used for T cells, and two panels were used for monocytes. Data were collected using a BD LSRII flow cytometer and analyzed with FCS Express software (DeNovo Software, Los Angeles, CA).

THP-1 monocytic cell line was cultured in complete RPMI 1640 medium supplemented with 10% FBS and 1% P/S. In order to induce a macrophage-like phenotype, cells were plated in 24-well plates (0.25 × 10⁶ cells/well) with a medium containing 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) (Sigma) and allowed to differentiate for 48h. To obtain macrophages at a resting state, adherent cells were cultured in a medium without PMA for an additional 24 h period. As a control, and mimicking a pro-inflammatory environment, THP-1 cells were stimulated with Interferon gamma (INF-γ, 20 ng/mL), and consequently a M1 polarization was promoted; macrophages were stimulated with interleukine-10 (IL-10, 10 ng/mL) to induce anti-inflammatory M2 macrophages and incubated for 3 additional days. Differentiated macrophages were incubated with the secretome of hDFMSC culture for 21 days within Coll/nanoHA scaffolds with or without OPN-Fb gel. Cells were harvested with 5mM EDTA-PBS and centrifuged at 300× g for 5 min at 4 °C, re-suspended in FACS buffer (PBS 1×, 2% FBS, 0.01% sodium azide), and immunostained with anti-CD86-FITC (Immunotools) and anti-CD163-PE (BD Biosciences Pharmingen, San Diego, CA, USA) were used as M1 and M2 specific markers, respectively, for 20 min at 4 °C in the dark.

To detect macrophage surface markers, anti-CD163-PE (BD Biosciences, USA) and anti-CD11b-APC (eBioscience, USA) antibodies were used to stain cells at 4 °C for 30 min. Isotype controls were run in parallel. After washing, a BD Accuri C6 flow cytometer (BD Biosciences) was used to determine the proportion of CD11b⁺CD163⁺ macrophages. To detect cell cycle arrest, glioma cells were collected 48 h after transfection and fixed with ice-cold 70% ethanol overnight. Then, the cells were incubated with 0.5 mL of propidium iodide (BD Biosciences) in the dark at room temperature for 15 min and were finally analyzed with a BD Accuri C6 flow cytometer (BD Biosciences) and ModFit software (Verity Software House, USA). An Annexin V-FITC apoptosis detection kit (BD Biosciences) was utilized to evaluate apoptosis according to the manufacturer’s instructions. In brief, cells were stained with Annexin V-FITC and propidium iodide. After incubation at room temperature in the dark for 15 min, apoptosis was quantified using a BD Accuri C6 flow cytometer (BD Biosciences) within 1 h of staining.

Collected 200 μl BM specimens from MM patients and HC, respectively. Subsequently, labeling the specimens with anti‐CD3‐PerCP, anti‐CD8‐FITC, and anti‐PD‐1‐PE antibodies. Meanwhile, another group was labeled with anti‐CD14‐PerCP, anti‐CD68‐FITC, anti‐CD163‐PE, and anti‐CD200R‐APC and anti‐PD‐L1 antibodies (BD Biosciences). Eventually, the expression of PD‐1 on CD8 + T cells and PD‐L1 on TAMs was detected by flow cytometry (FCM) (Beckman CytoFLEX), and the data were analyzed by Cell QuestTMPro 4.0.2 software.

Cells were stained with anti-CD163-PE (BD Biosciences, CA, USA) at 4 °C for 20 min, washed twice with FACS buffer, and then fixed with 4% paraformaldehyde for 20 min at 4 °C. Cells were washed twice with PBS and permeabilized with 0.3% Triton X-100 in PBS at 4 °C for 10 min. After the cells were washed twice with PBS, they were blocked with 5% BSA in PBS at 4 °C for 30 min. Anti-Wnt5a-FITC (Aviva Systems Biology, CA, USA) and anti-CD68-perCP (BioLegend, CA, USA) were added in blocking buffer and incubated overnight at 4 °C. Cells were washed twice with PBS and incubated at room temperature for 60 min. Cells were washed twice with PBS, and DAPI was added and incubated at room temperature for 10 min. Images were collected on a Nikon A1R confocal microscope.