The supernatant of FBs stimulated by arecoline was used as the conditioned medium for the experimental group whereas the supernatant of FBs without arecoline stimulation was utilized as the conditioned medium for the control group. After 48 h of treatment, the samples were analysed by flow cytometry. Cell density was adjusted to 1 × 107 cells/mL, then macrophages incubated with FITC anti‐human CD14, PE anti‐human CD68, APC anti‐human CD209, PE anti‐human CD163, Cy5 anti‐human CD206 (BioLegend). Finally, the cells were detected with flow cytometry (Cyto‐Flex Beckman).
Pe anti human cd68
Manufactured by BioLegend
Sourced in United States
PE anti-human CD68 is a fluorescent-conjugated antibody that binds to the CD68 antigen expressed on human macrophages and monocytes. It is used in flow cytometry applications to identify and quantify these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Fc block, by BD (2 mentions)
Accuri c6, by BD (2 mentions)
Viability stain, by Thermo Fisher Scientific (2 mentions)
Live dead fixable green, by Thermo Fisher Scientific (2 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions)
Foxp3 transcription factor staining buffer kit, by Cytek Biosciences (2 mentions)
Apc faser kit, by Miltenyi Biotec (2 mentions)
Fitc anti human cd14, by BioLegend (1 mentions)
Pe anti human cd163, by BioLegend (1 mentions)
Beckman cytoflex, by Beckman Coulter (1 mentions)
Apc anti human cd209, by BioLegend (1 mentions)
True phos perm buffer, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Fitc anti human cd206, by BioLegend (1 mentions)
Apc anti human cd124 il 4rα, by BioLegend (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti human il 10 apc, by BioLegend (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
6 protocols using pe anti human cd68
1
Macrophage Phenotyping in FB Arecoline Stimulation
2
Characterizing Macrophage Phenotypes by Flow Cytometry
3
Evaluating RUNX1 Impact on M2 Macrophage Polarization
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To evaluate the effect of RUNX1 on M2 polarization of TAMs, macrophages were washed, trypsin digested or scraped off, and processed into single-cell suspensions after the supernatant was collected for cytokine detection. Then the cell samples were labeled with antibodies as follows: PE anti-human CD68 (333808, BioLegend), FITC anti-human‐CD206 (321104, BioLegend) and APC anti-human‐CD200R (329308, BioLegend). After membrane staining of CD206 and CD200R, intracellular staining for CD68 was performed with Fix/Perm buffer reagents (421002, 420801, BioLegend) according to the manufacturer’s procedure. M2 macrophages were identified as CD68+CD206+ cells, CD68+CD200R+ cells. For the rescue experiments, THP-1 was stimulated with CMs derived from HCT116 NC/RUNX1OE cells along with or without GDC-0449. The cell samples were stained with the above protocol. The cells were measured using a FACS Calibur flow cytometer (BD FACSAria III, BD Biosciences), and the acquired data was analyzed by FlowJo software (Tree Star, San Carlos, Calif., USA).
4
Macrophage Phenotyping and Phagocytosis Analysis
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Macrophages were incubated with Fc block (BD Biosciences, Franklin Lakes, NJ, U.S.) for 10 minutes at 4°C, then incubated with primary-conjugated antibodies (Biolegend, San Diego, CA, U.S.) and viability stain (Invitrogen, Carlsbad, CA, U.S.) for 15 minutes at 4°C. Antibodies used to stain for IL-4Rα were APC anti-human CD124 (IL-4Rα) (Biolegend, clone G077F6, 1:100) and Live/Dead Fixable Green (Invitrogen, 1:200). To amplify IL-4Rα signal, APC FASER kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for two cycles, according to kit instructions. viability stain used to stain macrophages for phagocytosis was Live/Dead Fixable Aqua (Invitrogen, 1:200).
Samples were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences, San Diego, CA, U.S.), then incubated with primaryconjugated antibodies for 45 minutes at 4°C. PE anti-human CD68 (Biolegend, clone Y1/82A, 1:100) was used to stain macrophages for phagocytosis. Data was measured on the Accuri C6 (BD) or LSR II (BD) flow cytometer and analyzed using FlowJo v10 software (BD).
Samples were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences, San Diego, CA, U.S.), then incubated with primaryconjugated antibodies for 45 minutes at 4°C. PE anti-human CD68 (Biolegend, clone Y1/82A, 1:100) was used to stain macrophages for phagocytosis. Data was measured on the Accuri C6 (BD) or LSR II (BD) flow cytometer and analyzed using FlowJo v10 software (BD).
5
Intracellular Cytokine Staining Assay
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For intracellular staining, cells were stimulated with 25 ng/mL phorbol 12-myristate 13-acetate and 250 ng/mL ionomycin (both from Sigma-Aldrich, St. Louis, MO, USA) for 4 h in the presence of Golgi-Stop (BD Biosciences, San Diego, CA, USA). PBMCs were stained with surface anti-human CD11c (BV510; BD Biosciences; 563026) and anti-human PD-L1 (FITC; Biolegend San Diego, USA; 393606) antibodies. The cells were permeabilized and fixed with CytoPerm/CytoFix (BD Biosciences) in accordance with the manufacturer’s instructions. After fixation and permeabilization, cells were stained with anti-human CD68 (PE; Biolegend; 12–0689–42) and anti-human IL-10 (APC; Biolegend; 506807). Flow cytometry was performed using CytoFLEX (Beckman Coulter, Fullerton, CA, USA).
6
Flow Cytometry Macrophage Activation and Tumor Cell Apoptosis
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Macrophages were collected and blocked with 3% bovine serum albumin (BSA) for 45 min, and incubated with anti-human CD86-PE (305406, BioLegend, USA, 1:200), anti-human CD206-APC (321109, BioLegend, USA, 1:200), or anti-human CD68-PE (333808, BioLegend, USA, 1:200) according to the manufacturers’ instructions. Tumor cells were collected and incubated with Annexin V-APC/PI cell death detection kit (abs50001, Absin, China, 1:50) according to the manufacturers’ instructions for 15 min. For each sample, at least 1 × 104 cells were analyzed with the Beckman CytoFLEX flow cytometry. Apoptosis rate (apoptosis%= early apoptotic cells (Upper right quadrant) + late apoptotic cells (Right lower quadrant).
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