Cells were collected and washed with FACS buffer twice, followed by incubation with anti-mouse CD16/32 (Fc blocker, #553141, BD Biosciences) for 5 min at 4 °C. Then, cells were stained with anti-mouse CD3e (APC, #553066, BD Biosciences), CD4 (PE, #553652, BD Biosciences), CD8 (BV605, #100744, BioLegend), CD69 (PECY7, #104511, BioLegend), CTLA4 (PE, #553720, BD Biosciences), PD-1 (FITC, #135213, BioLegend), CD8 (FITC, #100705, BioLegend), CD4 (APCCY7, # 100413, BioLegend), or CD8 (PECY7, #100721, BioLegend) for 30 min at 4 °C. Each sample was washed with FACS buffer twice and resuspended in 0.5 ml FACS buffer. DAPI was used to exclude dead cells. Fluorescent cells were detected with CytoFLEX LX Flow Cytometer (Beckman).
Anti mouse cd16 32 fc blocker
Manufactured by BD
Sourced in United States
The Anti-mouse CD16/32 Fc blocker is a laboratory reagent used to block Fc receptors on mouse cells. It is designed to prevent non-specific binding of antibodies to Fc receptors, which can interfere with the accurate detection and analysis of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (3 mentions)
Fixation permeabilization buffer kit, by Thermo Fisher Scientific (3 mentions)
Anti cd45, by Thermo Fisher Scientific (2 mentions)
Anti cd3, by Thermo Fisher Scientific (2 mentions)
Anti cd4, by Thermo Fisher Scientific (2 mentions)
Anti cd11b, by Thermo Fisher Scientific (2 mentions)
Anti siglec f, by Thermo Fisher Scientific (2 mentions)
Anti f4 80, by Thermo Fisher Scientific (2 mentions)
Anti nk1, by Thermo Fisher Scientific (2 mentions)
Anti cd8, by Thermo Fisher Scientific (2 mentions)
Anti ly6c, by Thermo Fisher Scientific (2 mentions)
Anti cd44, by Thermo Fisher Scientific (2 mentions)
Anti cd62l, by Thermo Fisher Scientific (2 mentions)
Cytoflex lx flow cytometer, by Beckman Coulter (1 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions)
Apc cy7, by BioLegend (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Anti cd45, by BD (1 mentions)
Anti cd44, by BD (1 mentions)
Anti cd69, by BD (1 mentions)
6 protocols using anti mouse cd16 32 fc blocker
1
Multiparameter Flow Cytometry Analysis
2
Comprehensive Flow Cytometry Analysis of Immune Cells
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Hepatic and adipose immune cells were pre‐incubated with anti‐mouse CD16/32 Fc blocker (BD Pharmingen, USA), followed by staining with the Live/Dead marker anti‐FVD‐APC‐Cy7 (all supplied by eBioscience, San Diego, CA, USA). The fluorochrome‐conjugated antibodies used in this study were anti‐CD45, anti‐CD3, anti‐NK1.1, anti‐CD4, anti‐CD8, anti‐CD44, anti‐CD62L, anti‐CD11b, anti‐F4/80, anti‐Ly6C, anti‐Ly6C, and anti‐Siglec‐F (all supplied by eBioscience, San Diego, CA, USA). Liver mononuclear cells were stimulated with phorbol‐myristate acetate/ionomycin/brefeldin A/monensin for 5 hr in vitro. The cells were fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA). The permeabilized cells were washed with FACS buffer and resuspended in 1% formaldehyde and stained for intracellular cytokines with anti‐IFN‐γ‐PE‐Cy7, anti‐TNF‐α‐APC, and anti‐IL‐17A‐APC fluorochrome‐conjugated antibodies. Stained cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA).
3
Phenotypic Profiling of Liver Immune Cells
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Isolated liver MNCs were resuspended in DPBS containing 0.5% BSA and 0.05% sodium azide. After washing, the cells were pre-incubated with anti-mouse CD16/32 Fc blocker (BD Pharmingen, USA) prior to treatment with antibodies to block non-specific reactions. The cells were stained with fluorescence-conjugated anti-CD45, anti-CD3e, anti-CD4, anti-CD8, anti-CD11b, anti-CD25, anti-CD44, anti-CD69, anti-Ly6C, anti-Ly6G, and anti-F4/80 antibodies (BD Pharmingen, USA). The stained cells were analyzed using a BD™ LSR II Flow Cytometer (BD Pharmingen, USA) and the FlowJo software (Tree Star, Ashland, OR, USA). PE-conjugated anti-IFN-γ and TNF-α antibodies (BD Bioscience, USA) were used for intracellular staining. For intracellular cytokine staining, the cells were re-stimulated with phorbol-myristate acetate/ionomycin for 1 hour in addition to brefeldin A for 5 hours and then the cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Pharmingen, USA).
4
Comprehensive Immune Cell Profiling
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PBMCs were pre-incubated with an anti-mouse CD16/32 Fc blocker (BD Pharmingen, USA), followed by anti-FVD-APC-Cy7 (all supplied by eBioscience, San Diego, CA, USA) to exclude dead cells. After washing with FACS staining buffer, cells were treated with fluorochrome-conjugated monoclonal antibodies for 40 min at 4°C. The monoclonal antibodies used in this study were as follows: anti-CD3-PerCP-Cy5.5, anti-CD3-PE-Cy7, anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD45RA-FITC, anti-CD45RO-PE-Cy7, anti-CD57-FITC, anti-TCR gamma/delta-FITC, fixable viability dye-APC-Cy7, anti-interferon (IFN)-γ-PE-Cy7, and anti-tumor necrosis factor (TNF)-α-APC (all supplied by eBioscience, San Diego, CA, USA). For intracellular staining, surface-stained cells were stimulated with phorbol-myristate acetate/ionomycin/brefeldin A/monensin for 5 h, and then fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA). The permeabilized cells were washed and resuspended in 1% formaldehyde and stained with anti-IFN-γ-PE-Cy7 and anti-TNF-α-APC. Multicolor flow cytometry was performed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed by FlowJo V10 software (FlowJo, LLC, Ashland, OR, USA).
5
Peptide-Loaded MHC Class I Tetramer Staining
6
Characterization of Hepatic Tumor Immune Cells
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Hepatic tumorous immune cells were preincubated with anti-mouse CD16/32 Fc blocker (BD Pharmingen, San Diego, CA, USA), followed by staining with the Live/Dead marker FVD-APC-Cy7 (all supplied by eBioscience, San Diego, CA, USA). The fluorochrome-conjugated antibodies used in this study were anti-CD45, anti-CD3, anti-NK1.1, anti-CD4, anti-CD8, anti-CD44, anti-CD62L, anti-CD279, anti-CD11b, anti-F4/80, anti-Ly6C, anti-Ly6C, anti‐Siglec-F, anti-MCL-1, and anti-BCL2 (all supplied by eBioscience). Hepatic tumor immune cells were stimulated with phorbol-myristate acetate/ionomycin/brefeldin A/monensin for 5 hours in vitro. Cells were fixed and permeabilized using the Fixation/Permeabilization Buffer kit (eBioscience), washed with FACS buffer, resuspended in 1% formaldehyde, and stained with anti‐IFN-γ-APC. Stained cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and the data were analyzed using the FlowJo software (FlowJo, LLC, Ashland, OR, USA). The antibodies used are listed in online supplemental table S3 .
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