Dox-inducible SUZ12-expressing cell, T265, were seeded then treated with Dox (0.5 μg/mL) the following day. Two million cells treated with or without Dox were harvested and washed once with flow staining buffer (FSB, PBS supplemented with 2% FBS and 1 mM EDTA) and blocked with anti-Fc receptor (Biolegend) at room temperature for 5 min. One million cells from each condition were stained with PE anti-human HLA-A,B,C or IgG isotype and the fixable viability dye eFluor 506, which were diluted with FSB at room temperature for 12 min. A separate half million cells were incubated on a heat block at 65°C for 1 min to kill the cells, then mixed with the remaining half million cells and stained with the fixable viability dye eFluor 506 alone for compensation. A separate million cells were stained with PE anti-human HLA-A,B,C alone for compensation. Post staining, cells were washed twice with FSB followed by resuspension in 300 μL of FSB and ready for flow cytometry. The LSRFortessa (BD, Franklin Lakes, NJ) flow cytometer was used to assess the fluorescent intensity of MPNST cells stained for human MHC I molecules. Cells were gated to retain live, single cells for downstream analysis of the protein of interest in FlowJo software (BD). The stacked intensity of HLA-A,B,C was visualized in FlowJo.
Anti fc receptor
Manufactured by BioLegend
Sourced in United States
The Anti-Fc receptor is a laboratory equipment product that functions as a tool for studying Fc receptor biology. It is designed to specifically bind to Fc receptors, which are important cell-surface proteins involved in immune cell signaling and function. The Anti-Fc receptor provides a means to investigate Fc receptor interactions, expression, and cellular responses.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
True stain monocyte blocker, by BioLegend (1 mentions)
Cytexpert software v 2, by Beckman Coulter (1 mentions)
Zombie yellow, by BioLegend (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Alexa fluor 647 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 system, by BD (1 mentions)
Fitc labeled anti mouse cd11b, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
5 protocols using anti fc receptor
1
Assessing MHC I Expression in Dox-Induced SUZ12 Cells
2
Monocyte Subset Analysis by Flow Cytometry
3
Assessing MHC I Expression in Dox-Induced SUZ12 Cells
4
Brain cell staining and analysis
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For brain cells staining, the Gca‐Cre‐EGFP mice were first injected with 2 ug of phycoerythrin (PE)–conjugated anti‐CD45 antibody as previously described.[54 (link)
] After 10 min, mice were euthanized. Brains were infused, minced and digested for 30 min with 1.5 mg mL−1 collagenase IV and 0.2 mg mL−1 DNase I at 37 °C. After centrifugation and washing, the pellet was first incubated with an anti‐Fc receptor (Biolegend, San Diego, CA) to reduce nonspecific binding of antibodies, followed by incubation with the indicated antibodies for 20–30 min at 4 °C. Then, cells were stained with PE‐CY7‐CD45 and BV510‐CD11b (Biolegend, San Diego, USA) for 30 min at 4 °C. For CCR10 staining, obtained bone marrow cells were stained with the CCR10 antibody (Proteintech, Rosemont, USA) for 1 h at 4 °C. The cells were further incubated with Alexa fluor 647‐anti rabbit IgG (Invitrogen, Carlsbad, USA) and APC‐Cy7‐CD45, BV510‐CD11b, PE‐Cy7‐Ly6C, PE‐Ly6G (Biolegend, San Diego, USA) for 30 min at 4 °C. All antibodies were diluted according to the manual from the manufacturer’ s website. Stained cells were collected by BD FACSCanto II system and analyzed by FlowJo V10. Dead cells and doublets were removed by FSC‐A/FSC‐H gating. Data were analyzed by FlowJo V10 (BD Biosciences, San Jose, USA).
] After 10 min, mice were euthanized. Brains were infused, minced and digested for 30 min with 1.5 mg mL−1 collagenase IV and 0.2 mg mL−1 DNase I at 37 °C. After centrifugation and washing, the pellet was first incubated with an anti‐Fc receptor (Biolegend, San Diego, CA) to reduce nonspecific binding of antibodies, followed by incubation with the indicated antibodies for 20–30 min at 4 °C. Then, cells were stained with PE‐CY7‐CD45 and BV510‐CD11b (Biolegend, San Diego, USA) for 30 min at 4 °C. For CCR10 staining, obtained bone marrow cells were stained with the CCR10 antibody (Proteintech, Rosemont, USA) for 1 h at 4 °C. The cells were further incubated with Alexa fluor 647‐anti rabbit IgG (Invitrogen, Carlsbad, USA) and APC‐Cy7‐CD45, BV510‐CD11b, PE‐Cy7‐Ly6C, PE‐Ly6G (Biolegend, San Diego, USA) for 30 min at 4 °C. All antibodies were diluted according to the manual from the manufacturer’ s website. Stained cells were collected by BD FACSCanto II system and analyzed by FlowJo V10. Dead cells and doublets were removed by FSC‐A/FSC‐H gating. Data were analyzed by FlowJo V10 (BD Biosciences, San Jose, USA).
5
Flow Cytometric Characterization of BM-MSCs
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BM-MSCs were harvested and washed with PBS containing 2% FBS (Staining buffer) once. Cells were pre-blocked with anti-Fc receptor III/II monoclonal antibody (mAb) (clone 2.4G2) (Biolegend, San Diego, CA, USA) for 5 min and then cells were stained with FITC labeled anti-mouse CD29, CD44, CD90, CD34 and CD45 or corresponding isotype control antibody (Biolegend) on ice for 30 min. After wash with staining buffer for three times, the samples were analyzed by BD FACSCalibur flow cytometer and data was analyzed using FlowJo. In certain experiment, heart cells were pre-blocked with anti-Fc receptor III/II monoclonal antibody (mAb) and stained with APC labeled anti-mouse CD68 and FITC labeled anti-mouse CD11b (Biolegend).
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