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Anti mouse cd16 cd32 clone 93

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The Anti-mouse CD16/CD32 (clone 93) is a laboratory reagent used for the detection of mouse CD16 and CD32 cell surface antigens. It is commonly used in flow cytometry and other immunoassays to block Fc receptor-mediated binding of antibodies.

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Lab products found in correlation

Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (3 mentions) Versacomp antibody capture bead kit, by Beckman Coulter (2 mentions) Gallios flow cytometer, by Beckman Coulter (2 mentions) Suramin, by Merck Group (2 mentions) Carbenoxolone, by Merck Group (2 mentions) Tosufloxacin, by Merck Group (2 mentions) Difloxacin, by Merck Group (2 mentions) Purifiednucleotides, by Merck Group (2 mentions) Recombinant apyrase, by New England Biolabs (2 mentions) Annexin 5 fitc, by Thermo Fisher Scientific (2 mentions) Levofloxacin, by Merck Group (2 mentions) Blebbistatin, by Merck Group (2 mentions) Probenecid, by Merck Group (2 mentions) Anti fas clone ch11, by Merck Group (2 mentions) Cd4 pe cy7 clone rm4 5, by Thermo Fisher Scientific (2 mentions) 7 aadand to pro 3, by Thermo Fisher Scientific (2 mentions) Cd8a pe clone53 6.7, by Thermo Fisher Scientific (2 mentions) Y 27632, by Merck Group (2 mentions) Cytochalasin d, by Merck Group (2 mentions) Ciprofloxacin, by Merck Group (2 mentions)

16 protocols using anti mouse cd16 cd32 clone 93

1

Immune cell identification protocol

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The following antibodies were used for identification of immune cells: anti-mouse CD16/CD32 clone 93 (eBioscience), Gr1-FITC (Ly-66) clone RB6-8C5 (eBioscience), SiglecF-PE clone ES22-10D8 (Miltenyi Biotec, Bergisch Gladbach, Germany), CD11b–peridinin-chlorophyll-protein complex (PerCP)–Cy5.5 (eBioscience), F4/80-APC (BD Biosciences), CD8a-PerCp-Cy5.5 clone 53-6.7 (eBioscience), CD4-FITC clone RM4-4 (eBioscience), MHCII clone M5/114.15.2 FITC (eBioscience), CD11c PE-Cy7 clone HL3 (eBioscience), and LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific). Cells were stained for 30 min at room temperature and washed with PBS; LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) was used for 10 min to exclude dead cells. Cells were washed and analyzed by using flow cytometry with the FACS Canto II (BD Biosciences).
Teufelberger A.R., Van Nevel S., Hulpiau P., Nordengrün M., Savvides S.N., De Graeve S., Akula S., Holtappels G., De Ruyck N., Declercq W., Vandenabeele P., Hellman L., Bröker B.M., Krysko D.V., Bachert C, & Krysko O. (2020). Mouse Strain-Dependent Difference Toward the Staphylococcus aureus Allergen Serine Protease-Like Protein D Reveals a Novel Regulator of IL-33. Frontiers in Immunology, 11, 582044.
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2

Single-Cell Immunophenotyping by Flow Cytometry

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The single-cell suspension samples were blocked in anti-mouse CD16/CD32 (clone 93; eBioscience) at 1:200 on a rocker for 15 min at 4 °C. Next, fix viability dye (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit; 1:200; Invitrogen) and conjugated antibodies were added (see below) to the single cell suspension. Cells were incubated in the dark at 4 °C for 60 min. Cells were then resuspended in FA3 buffer and ran on the Gallios Flow Cytometer (Beckman Coulter). For compensation, single-stained beads (VersaComp Antibody Capture Bead Kit; Beckman Coulter) and a single-stained cell-mix of all samples analyzed were used. Flow cytometry was analyzed using Kaluza Analysis Software (v2.0, Beckman Coulter). Compensation was first performed on the single-stained bead controls and then confirmed using the single-stained cell mixture.
Kleczko E.K., Hinz T.K., Nguyen T.T., Gurule N.J., Navarro A., Le A.T., Johnson A.M., Kwak J., Polhac D.I., Clambey E.T., Weiser-Evans M., Merrick D.T., Yang M.C., Patil T., Schenk E.L., Heasley L.E, & Nemenoff R.A. (2023). Durable responses to alectinib in murine models of EML4-ALK lung cancer requires adaptive immunity. NPJ Precision Oncology, 7, 15.
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3

Phenotypic Analysis of Dendritic Cells

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Cells were collected in phosphate buffered saline (PBS) and pre-incubated with Fc receptor blocking anti-mouse CD16/CD32 (clone 93) or human Fc receptor binding inhibitor (eBioscience Inc., USA). A blue fluorescent amine-reactive dye (Invitrogen, USA) was used as live/dead cell marker. Murine cells were decorated with anti-mouse CD80 (clone 16-10A1, APC-conjugated), CD86 (clone GL1, PE-conjugated), I-Ab (clone AF6-120.1, FITC-conjugated), CD11c (clone N418, PE-Cy7-conjugated); human cells with anti-human CD40 (clone 5C3, PE-conjugated), CD54 (clone HA58, APC-conjugated), CD80 (clone 2D10, APC-conjugated), CD83 (clone HB15e, PE-conjugated), CD86 (clone IT2.2, PE-Cy7-conjugated), CD11c (clone 3.9, Brilliant Violet 711-conjugated) (BioLegend, USA). Flow cytometry analysis was performed using an LSR-II and the software FACSDiva (BD Bioscience, USA) and FlowJo Mac v9.6 (Tree Star, Inc., USA). Forward and sideward scatter gating was used to restrict fluorescence analysis to intact single cells only. Live cells were gated based on their lower blue fluorescence emission of the live/dead cell marker and DCs were identified by their enhanced surface expression of CD11c. Activated cells were identified by gating for cell populations with enhanced fluorescence intensity indicative of the antibody-detected surface molecules.
Škrnjug I., Guzmán C.A, & Ruecker C. (2014). Cyclic GMP-AMP Displays Mucosal Adjuvant Activity in Mice. PLoS ONE, 9(10), e110150.
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4

Flow Cytometric Immunophenotyping of Mononuclear Cells

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Mononuclear cells were suspended in 100 μL FACS buffer and blocked with 1μL anti-mouse CD16/CD32 (clone 93, Catalog No. 14-0161-85, eBioscience) for 20 min. The cells were immunostained for 30 min in the dark with 1μL of each of the following antibodies against: CD11b (clone M1/70, PerCP 5.5, Catalog No. 45-0112-82, eBioscience), CD45 (clone 30-F11, FITC, Catalog No. 11-0451-81, eBioscience), Ly6G Gr-1 (clone 1A8-Ly6G, PE-eFluor 610, Catalog No. AB_2574679, eBioscience), CD36 (clone CRF D-2712, PE, Catalog No. 562702 BD Pharmingen; clone 72-1,PE, Catalog No. 12-0361-82, eBioscience), IgG Isotype control (clone M18-254, PE, Catalog No. 562141, BD Pharmingen; clone eBR2a, PE, Catalog No. 12-4321-82, eBioscience), and fixable viability dye (eFluor 506, Catalog No. 65-0866-14, eBioscience). 50 μL Absolute counting beads (Catalog No. C36950, Life Technologies, Carlsbad, CA) were used for cell counting. After washing, the cells were resuspended in 100 μL of FACS buffer and 100 μL 2% PFA. Data were acquired using a BD FACS ARIAIII and were analyzed using FlowJo v10 software (FlowJo). BMDM cell death rate was calculated as (100%- %viability).
Kanoke A., Nishijima Y., Ljungberg M., Omodaka S., Yang S.Y., Wong S., Rabiller G., Tominaga T., Hsieh C.L, & Liu J. (2020). The effect of type 2 diabetes on CD36 expression and the uptake of oxLDL Diabetes affects CD36 and oxLDL uptake. Experimental neurology, 334, 113461.
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5

Comprehensive Pharmacological Tools for Research

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Library of Pharmacologically Active Compounds (LOPAC1280TM),
trovafloxacin, ciprofloxacin, levofloxacin, difloxacin, tosufloxacin,
carbenoxolone, probenecid, Y-27632, blebbistatin, cytochalasin D, purified
nucleotides, suramin and dexamethasone were obtained from Sigma-Aldrich. 7-AAD
and TO-PRO-3 were purchased from Invitrogen. annexin V-FITC, CD8a-PE (clone
53-6.7), CD4-PE-Cy7 (clone RM4-5) and anti-mouse CD16/CD32 (clone 93) were
obtained for eBioscience. Other reagents were obtained as follows: anti-Fas
(clone CH11, Millipore), z-VAD-FMK (Enzo Life Sciences), Q-VD-OPH (SM
Biochemicals) and recombinant apyrase (New England Biolabs).
Poon I.K., Chiu Y.H., Armstrong A.J., Kinchen J.M., Juncadella I.J., Bayliss D.A, & Ravichandran K.S. (2014). Unexpected link between an antibiotic, pannexin channels, and apoptosis. Nature, 507(7492), 329-334.
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6

Immunophenotyping of Mouse Lung Cells

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Lung cells were stained after FcγRII/III blocking with anti‐mouse CD16/CD32 (clone 93; eBioscience, California, United States). The following anti‐mouse mAbs purchased from Becton, Dickinson and Company (BD, New Jersey, United States), BioLegend or eBioscience were used: CD45 (30‐F11), CD11c (N418), F4/80 (BM8), CD4 (RM4‐5), CD25 (PC61.5), CXCR3 (CXCR3‐173), CXCR5 (SPRCL5), CCR2 (SA203G11), CCR4 (2G12), CCR5(HM‐CCR5), Foxp3 (FJK‐16s), T‐bet (eBio4B10), Ki67 (SolA15), LAG3 (C9B7W), ICOS (15F9), CTLA (UC10‐4B9), LAP (TW7‐16B4) and IL‐10 (JES5‐16E3). For the intracellular staining of Foxp3, T‐bet, Ki67 and IL‐10, cells were surface‐stained with CD4 and followed by permeabilisation with Foxp3 staining kit (eBiosciences). Then, the cells were stained with Foxp3, Ki67, IL‐10 and T‐bet. FACSCalibur or FACS Fortessa (BD) were employed to collect the data. Data were then analysed with FlowJo software.
Li W., Chen W., Huang S., Yao G., Tang X, & Sun L. (2020). Mesenchymal stem cells prevent overwhelming inflammation and reduce infection severity via recruiting CXCR3+ regulatory T cells. Clinical & Translational Immunology, 9(10), e1181.
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7

Comprehensive Immune Cell Profiling

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Mouse splenocytes were stained with Zombie NIR in PBS (Biolegend, Cat# 423106) at room temperature for 20 minutes. The cells were incubated with anti-mouse CD16/CD32 (clone 93; eBiosciences) in PBS containing 2% dry milk to block FcγRII/III at 4 °C for 30 min. Surface staining was performed with anti-CD3e (clone 145-2C11), anti-CD4 (clone rm4–5), anti-CD8α (clone 53-6.7), anti-CD19 (clone 6D5), anti-B220 (clone RA3–6B2), anti-CD11c (clone N418), anti-CD11b (clone M1/70), and anti-ly6G (clone 1A8) purchased from Biolegend (San Diego, CA).
Xia L., Wu J., Pattaradilokrat S., Tumas K., He X., Peng Y.C., Huang R., Myers T.G., Long C.A., Wang R, & Su X.Z. (2018). Detection of host pathways universally inhibited after Plasmodium yoelii infection for immune intervention. Scientific Reports, 8, 15280.
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8

Quantification of Alveolar Macrophages and Neutrophils

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BALF and lung cells were stained at 4°C in RPMI 1640 medium containing 1% FBS after FcγRII/III blocking with anti-mouse CD16/CD32 (clone 93; eBioscience). Surface staining was performed with antibodies purchased from eBioscience (anti-CD45; clone 30-F11; anti-Ly6G (Gr-1), clone RB6-8C5; anti-CD11c, clone N418; and anti-CD11b, clone M1/70). Alveolar macrophages (CD11b−CD11c+) and neutrophils (CD11b+Gr-1+) were analysed.
For intracellular staining, lung and spleen cells were stimulated with heat-killed bacteria at MOI (multiplicity of infection) 100 for 16 h at 37°C with Golgi block added on the last 5 hours. Cells were then surface-stained with anti-CD4 (clone RM4-5; eBioscience) and anti-γδTCR (clone GL-3; eBioscience) and followed by permeabilization with Cytofix-Cytoperm solution (BD Pharmingen). Then, the cells were stained with anti-IL-17A (clone 17B7; eBioscience) and anti-IFN-γ (clone XMG1.2; eBioscience). All samples were analysed with FACSCalibur. Data were analysed with FlowJo software.
Li W., Chen W., Huang S., Tang X., Yao G, & Sun L. (2019). Infection with Opportunistic Bacteria Triggers Severe Pulmonary Inflammation in Lupus-Prone Mice. Mediators of Inflammation, 2019, 1701367.
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9

Neutrophil and Monocyte Profiling in Murine Blood

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Peripheral blood was collected via cardiac puncture in 3.2 % sodium citrate. Cells were then incubated with anti–mouse CD16/CD32 (clone 93, eBioscience; dilution 1:100, 20 minutes at 4°C in PBS containing 5% FCS staining solution). Peripheral blood cells were then incubated with AF700-conjugated anti–mouse Ly6G (clone 1A8, BioLegend; for neutrophils) and BV421-conjugated anti–mouse CD115 (clone AFS98, BioLegend; for monocytes) antibodies (30 minutes at 4°C), and receptor expression was evaluated as described above.
Flak M.B., Koenis D.S., Sobrino A., Smith J., Pistorius K., Palmas F, & Dalli J. (2019). GPR101 mediates the pro-resolving actions of RvD5n-3 DPA in arthritis and infections. The Journal of Clinical Investigation, 130(1), 359-373.
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10

Flow Cytometry Analysis of Tumor-Infiltrating T Cells

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A single cell suspension was performed on EA1-NTC or EA1-shCfh-63 tumors. At sacrifice, the lungs were perfused with 5 mL of PBS/heparin (20U/mL; Sigma). The single cell suspension samples were blocked in anti-mouse CD16/CD32 (clone 93; eBioscience) at 1:200 on a rocker for 15 min at 4°C. Next, fix viability dye (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit; 1:200; Invitrogen; #L34966) and conjugated antibodies were added (see Antibody Panel section below) to the single cell suspension. Cells were incubated in the dark at 4°C for 60 minutes. Cells were then resuspended in FA3 buffer and ran on the Gallios Flow Cytometer (Beckman Coulter). For compensation, single-stained beads (VersaComp Antibody Capture Bead Kit; Beckman Coulter) and a single-stained cell-mix of all samples analyzed were used. Flow cytometry was analyzed using Kaluza Analysis Software (v2.0, Beckman Coulter). Compensation was first performed on the single-stained bead controls and then confirmed using the single-stained cell mixture.
T cell Antibody Panel: CD8-FITC (clone 53-6.7; 1:100; Invitrogen), MHCII-Dazzle (clone M5/111.15.2; 1:250; BioLegend), CD3-AF700 (clone 17A2; 1:100; BioLegend), CD4-APC/Cy7 (clone GK1.5; 1:200; BioLegend), CD45-eFlour450 (clone 30-F11; 1:100; Invitrogen), CD4-V500 (clone RM4-5; 1:200; BD Biosciences; used only for compensation).
Kleczko E.K., Poczobutt J.M., Navarro A.C., Laskowski J., Johnson A.M., Korpela S.P., Gurule N.J., Heasley L.E., Hopp K., Weiser-Evans M.C., Gottlin E.B., Bushey R.T., Campa M.J., Patz EF J.r., Thurman J.M, & Nemenoff R.A. (2023). Upregulation of complement proteins in lung cancer cells mediates tumor progression. Frontiers in Oncology, 12, 1045690.
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