The cells collected from the peritoneal fluid after 24 h of culture for adherence were fixed with a buffer containing paraformaldehyde (554722, BD Cytofix/Cytoperm USA), washed two times with 1× BD wash buffer (554723, BD Perm/Wash, USA), centrifuged at 350 × g for 5 min, and then the supernatant was discarded. For membrane cytokines, such as CD163 (anti-human CD163; 333606, BioLegend, USA) and CD86 (anti-human CD86; 374216, BioLegend, USA), cells were incubated in diluted primary antibody buffer in the dark for 20 min at about 26°C. For intracellular cytokines, such as CD68 (anti-human CD68; 333806, BioLegend, USA) perm buffer (554723, BD Perm/Wash, USA) was used for 15 min for permeabilization, and the supernatant was discarded after centrifuging at 350 × g for 5 min. Cells were incubated in diluted primary antibody buffer in the dark for 30 min at 4 °C, washed twice with wash buffer, and centrifuged at 350 × g for 5 min. The cells were then resuspended in perm buffer for FCM analysis.
Anti human cd163
Manufactured by BioLegend
Sourced in United States
Anti-human CD163 is a monoclonal antibody that specifically binds to the CD163 protein, a cell surface receptor expressed primarily on monocytes and macrophages. CD163 plays a role in the clearance of hemoglobin-haptoglobin complexes.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd86, by BioLegend (3 mentions)
Anti human cd68, by BioLegend (1 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
7 aad antibodies, by BioLegend (1 mentions)
Cell strainer, by BD (1 mentions)
Ab18528, by Abcam (1 mentions)
Tcs sp5 2 microscope, by Leica (1 mentions)
Cytospin 4 cytocentrifuge, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Dm2500 microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Antimouse cd206, by Thermo Fisher Scientific (1 mentions)
Anti human hla dr, by BioLegend (1 mentions)
Anti human cd80, by BioLegend (1 mentions)
Anti human cd16, by BioLegend (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
5 protocols using anti human cd163
1
Macrophage Phenotyping Protocol
2
Sorting CD163+CD14+ and CD163-CD14+ Cells
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CD163+CD14+ and CD163−CD14+ populations were sorted from mononuclear cells derived from MPE using Moflo XDP (Beckman) (n=6). In brief, cell clumps were removed by passing cell suspensions through 40 mm Cell Strainers (BD Biosciences). 1×108 mononuclear cells were stained with 20 μl of anti-human CD163, CD14 and 7-AAD antibodies (Biolegend) respectively. Then, cells were incubated in the dark for 15 min at 4 °C. Cells were resuspended with 1 ml of normal saline for sorting. The purities of sorted CD163+CD14+ and CD163−CD14+ cells were analyzed by FACS.
3
Multimodal Immunophenotyping of Tumor Samples
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For immunofluorescence staining of OCT frozen tumor tissue sections, serum and catalase blocking was performed by goat serum (ZSGB-Bio Company) and 0.3% H2O2 respectively. Then sections were incubated with anti-LAMP2a (Abcam ab18528), anti-F4/80 (Abcam ab16911), anti-human CD68 (Biolegend 333805), anti-human CD163 (Biolegend 333605), and corresponding secondary antibodies (Invitrogen, Abcam). After washing, DAPI (Beyotime Biotechnology) was stained and coverslips were mounted by Pro-Long Gold antifade reagent (Invitrogen). The staining sections were imaged by Leica TCS SP5 II microscope.
For staining of cell sliders, mouse primary cells were seeded in well-plates with glass slides placed in advance. Exfoliated cells from patients or sorted mouse cells from FACS were spotted in slides by Thermo Scientific Cytospin 4 Cytocentrifuge. The immunofluorescence staining procedures were consistent with tumor tissue sections above. The Giemsa staining was performed by commercial assay (BASO BA4007), imaged by Leica DM2500 microscope.
For TUNEL assays, the formalin treated mouse tumor tissues were sectioned by paraffin embedding. And the staining procedures followed manufacturer's protocol (Promega G7130), with images obtained by Leica DM2500 microscope.
For staining of cell sliders, mouse primary cells were seeded in well-plates with glass slides placed in advance. Exfoliated cells from patients or sorted mouse cells from FACS were spotted in slides by Thermo Scientific Cytospin 4 Cytocentrifuge. The immunofluorescence staining procedures were consistent with tumor tissue sections above. The Giemsa staining was performed by commercial assay (BASO BA4007), imaged by Leica DM2500 microscope.
For TUNEL assays, the formalin treated mouse tumor tissues were sectioned by paraffin embedding. And the staining procedures followed manufacturer's protocol (Promega G7130), with images obtained by Leica DM2500 microscope.
4
Macrophage Polarization Profiling by Flow Cytometry
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Macrophage polarization was detected by flow cytometry. Briefly, cells were incubated with the antibody conjugated with fluorescence in 100 μL of flow cytometry staining buffer (S1001, Multi Sciences) and were protected from light and incubated for 30 min at 20°C‐30°C. The data were analyzed with FlowJo‐V10 CL software. Each assay was replicated three times. Antibodies for flow cytometry: anti‐human CD86 (Cat#305419), anti‐human CD163 (Cat#333603), anti‐human CD14 (Cat#301803) were purchased from Biolegend (San Diego, CA, USA); anti‐mouse CD206 (Product#12‐2061‐80) was purchased from Invitrogen; anti‐mouse CD11c (70‐AM011C05‐100) and anti‐mouse F4/80 (AM04800201) were purchased from Multi Sciences.
5
Exploring Immune Profiles in Active and Inactive AOSD
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A total of 37 AOSD patients (27 active and 10 inactive AOSD patients) and 12 HCs were included for flow cytometry. The clinical information of AOSD patients was provided in the Additional file 1 : Table S1 and Additional file 1 : Table S2. Heparinized whole blood from AOSD patients and HCs were stained using following antibodies: anti-human CD14 (Biolegend, San Diego, CA, USA, catalog: 325,604), anti-human CD16 (Biolegend, clone:3G8, catalog: 302,012), anti-human CD80 (Biolegend, catalog: 305,221), anti-human CD86 (Biolegend, catalog: 305,431), anti-human CD163 (Biolegend, catalog: 333,608), anti-human HLA-DR (Biolegend, catalog: 307,653). All assays were performed by a FACS Canto II cytometer (BD). Data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
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