Anti cd16 cd32 clone 2.4g2

Manufactured by BD

Sourced in United States

Anti-CD16/CD32 (clone 2.4G2) is a laboratory reagent that binds to the Fc receptors CD16 and CD32 expressed on various immune cells. This product is commonly used in flow cytometry, cell-based assays, and other immunological applications to block Fc receptor-mediated interactions.

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Lab products found in correlation

Golgistop, by BD (4 mentions) Facscalibur, by BD (4 mentions) Cxp analysis software, by Beckman Coulter (3 mentions) Fc500 flow cytometer, by Beckman Coulter (3 mentions) Ecd conjugated anti cd8α clone 53 6.7, by Beckman Coulter (3 mentions) Fluorescently labeled antibodies, by BD (2 mentions) Pe cy7 conjugated anti cd62l clone mel 14, by Beckman Coulter (2 mentions) Lsrii flow cytometer, by BD (2 mentions) Cellquest software, by BD (2 mentions) Anti gr1 pe clone rb6 8c5, by BD (1 mentions) Centrifuge 5804 r, by Eppendorf (1 mentions) Facsaria 2 sorter, by BD (1 mentions) Pe anti cd4 clone gk1.5, by BD (1 mentions) Pe anti tcr γδ clone gl3, by BD (1 mentions) Anti cd45 percp clone 30 f11, by BD (1 mentions) Facsflow, by BD (1 mentions) Anti foxp3 clone fjk 16s, by Thermo Fisher Scientific (1 mentions) Anti f4 80 clone bm8, by Thermo Fisher Scientific (1 mentions) Anti cd11c clone hl3, by BD (1 mentions) Anti ly6c clone al 21, by BD (1 mentions)

21 protocols using anti cd16 cd32 clone 2.4g2

Bronchoalveolar Lavage and Neutrophil Analysis

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Mice were sacrificed with an intraperitoneal injection of sodium pentobarbital (20 mg/mice) and bronchoalveolar lavage was performed by cannulating the trachea and lavaging the lung 4× with 1ml of NaCl 0.9%. The broncho-alveolar lavage fluid (BALF) was centrifuged at 281g, 10 min, 4°C (Centrifuge 5804R, Eppendorf, Hamburg, Germany). Cells recovered in BAL were counted then fixed in 1.25% paraformaldehyde and analyzed with FACS calibur (BD Biosciences) using FlowJo software. Neutrophils were identified by labelling with anti-GR1-PE (clone RB6-8C5, BD Biosciences, Erembodegem, Belgium) and their number calculated in function of total BAL cells counted with a Burker cell chamber. Fc receptors were blocked with anti-CD16/CD32 (clone 2.4G2, BD Biosciences) to reduce nonspecific binding.

Rabolli V., Badissi A.A., Devosse R., Uwambayinema F., Yakoub Y., Palmai-Pallag M., Lebrun A., De Gussem V., Couillin I., Ryffel B., Marbaix E., Lison D, & Huaux F. (2014). The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Particle and Fibre Toxicology, 11, 69.

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Murine Hematological Characterization

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Erythrocyte (RBC), platelet (PLT) and total white blood cell (WBC) counts were determined in whole blood collected in EDTA of 6 week-old female and male mice using a VetABC hematology analyser. Blood and bone marrow leukocyte subsets percentages and cell death kinetics of bone marrow cells were determined using a 4-color FACS Calibur (BD Biosciences) using single cell suspensions blocked with anti-CD16/CD32 (clone 2.4G2) and stained for 30–40 min on ice with fluorescently labeled antibodies (1:200; BioLegend, BD Biosciences) as previously (Benarafa et al., 2011 (link)). Analysis was performed using FlowJo gating within nucleated live CD45⁺ cells as neutrophils (CD11b⁺/Ly6G⁺), monocytes (CD11b⁺/CD115⁺), B cells (CD45R/B220⁺), eosinophils (SSC^high/SiglecF⁺), NK cells (NK1.1⁺/CD3^neg) and T cells (CD3⁺). Apoptosis and necrosis of neutrophils and monocytes in vitro was determined by Annexin V-allophycocyanin and 7aminoactinomycin D (7AAD) labelling for 15min at room temperature prior to FACS analysis. Flow cytometry sorting of neutrophils (CD11b⁺/Ly6G⁺) was performed on single-cell suspensions of bone marrow leukocytes using a FACS Aria II sorter (BD Biosciences) at the flow cytometry core facility of the University of Bern.

Burgener S.S., Leborgne N.G., Snipas S.J., Salvesen G.S., Bird P.I, & Benarafa C. (2019). Cathepsin G inhibition by Serpinb1 and Serpinb6 prevent programmed necrosis in neutrophils and monocytes and reduce GSDMD-driven inflammation. Cell reports, 27(12), 3646-3656.e5.

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Multiparametric Flow Cytometry Analysis

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The following monoclonal antibodies were used for lymphocyte surface staining: Fluorescein isothiocyanate (FITC) anti-CD3 (clone 17.12), phycoerythrin (PE) anti-CD4 (clone GK1.5), phycoerythrin–cyanine 5 (PE-Cy5) anti-CD8 (clone 53-67), PE anti-CD19 (clone ID3), peridinin–chlorophyll-protein (PerCP) anti-CD45 (clone 30-F11), PE anti-TCR γδ (clone GL3), PE-Cy5 anti-TCR αβ (clone H57-597) and anti-CD16/CD32 (clone 2.4G2) (BD Pharmingen, Milan, Italy). Each antibody was previously titrated to determine the optimal concentration for maximal staining. The IELs (1 × 106 cells) were pre-incubated for 20 min with anti-CD16/CD32 to block Fc receptors. Cells were then washed and labelled with the appropriate mixture of antibodies for 30 min, centrifuged at 650× g and resuspended in FacsFlow (BD Biosciences, Milan, Italy). Flow cytometry analysis was performed using a FACSCalibur instrument (BD Biosciences). To exclude dead/dying cells and, therefore, non-specific antibody-binding cells, lymphocytes were gated according to forward and side scatter. The percentage of B and T lymphocytes was calculated on leukocyte (CD45+) gate, whereas the CD4+, CD8+ and CD4+CD8+ subsets, as well as αβ and γδ lymphocytes, were calculated on T lymphocyte (CD3+) gate. At least 10,000 events were acquired. Data were analysed using CellQuest software (BD Biosciences).

Carcea M., Turfani V., Narducci V., Durazzo A., Finamore A., Roselli M, & Rami R. (2019). Bread for the Aging Population: The Effect of a Functional Wheat–Lentil Bread on the Immune Function of Aged Mice. Foods, 8(10), 510.

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Multiparametric Flow Cytometry Analysis

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Flow cytometry analysis was performed by Attune Acoustic Focusing Cytometer (Life Technologies) using FlowJo software (Tree Star). For Intracellular cytokine staining, cells were stimulated with 20 ng/mL phorbol 12‐myristate 13‐acetate (Sigma) and 1 mmol/L ionomycin (Sigma) for 5 hour in the presence of a GolgiStop (BD Bioscience). The antibodies used were as follows; anti‐CD16/CD32 (clone 2.4G2; BD Bioscience), anti‐CD4 (clone H129.19; BD Bioscience), anti‐CD25 (clone PC61; BD Bioscience), anti‐CD103 (clone M290; BD Bioscience), anti‐GITR (clone DTA1; BD Bioscience), anti‐CTLA‐4 (clone UC10; BD Bioscience), anti‐Foxp3 (clone FJK‐16s; eBioscience), anti‐CD11c (clone HL3; BD Bioscience), anti‐CD80 (clone 16‐10A1; BD Bioscience), anti‐CD86 (clone GL1; BD Bioscience), anti‐CD49b (clone HMa2; BD Bioscience), anti‐LAG3 (clone C9B7W; BD Bioscience), anti‐CD11b (clone M1/70; BD Bioscience), anti‐Ly6C (clone AL‐21; BD Bioscience), anti‐CD115 (clone AFS98; eBioscience), anti‐F4/80 (clone BM8; eBioscience), anti‐CD206 (clone C068C2; BioLegend), anti‐IFNγ (clone XMG1.2; eBioscience), anti‐IL‐4 (clone BVD4‐1D11; eBioscience), anti‐IL‐10 (clone JES5‐16E3; eBioscience) and isotype‐matched control antibodies.

Kasahara K., Sasaki N., Yamashita T., Kita T., Yodoi K., Sasaki Y., Takeda M, & Hirata K. (2014). CD3 Antibody and IL‐2 Complex Combination Therapy Inhibits Atherosclerosis by Augmenting a Regulatory Immune Response. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 3(2), e000719.

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Peritoneal Mast Cell Characterization

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Peritoneal lavage was performed using 5 mL PBS supplemented with 1% heat-inactivated FBS on healthy female and male mice. Total cell numbers were evaluated by counting cells manually in a Neubauer chamber. Relative percentages of live mast cells were determined by flow cytometry; analysis was performed using FlowJo with single cell gating, dead cell exclusion using propidium iodide (PI) and, mast cells identified as FcεRI⁺, IgE⁺, and, c-kit⁺. Single cell suspensions blocked with anti-CD16/CD32 (clone 2.4G2) and stained for 30–40 min on ice with fluorescently labeled antibodies (BioLegend, BD Biosciences) were acquired using a 4-color FACS Calibur (BD Biosciences). Mast cells were permeabilized using the Foxp3 buffer (eBioscience) and stained using an anti-mouse granzyme A antibody (eBioscience, clone 3G8.5).

Burgener S.S., Brügger M., Leborgne N.G., Sollberger S., Basilico P., Kaufmann T., Bird P.I, & Benarafa C. (2021). Granule Leakage Induces Cell-Intrinsic, Granzyme B-Mediated Apoptosis in Mast Cells. Frontiers in Cell and Developmental Biology, 9, 630166.

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Multiparameter Flow Cytometry Analysis of Peritoneal Cells

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Peritoneal cells were resuspended in FACS Buffer (PBS 1x, 2mM EDTA, 0,5% BSA). Cell suspension was blocked with anti-CD16/CD32 (clone 2.4G2; BD Biosciences Pharmingen) for 10 min at 4°C. BD Cytofix/Cytoperm™ was used to fixation and permeabilization of the cells according to the manufactory instructions (Biosciences Pharmingen). The cells were stained with mouse anti-CD11b FITC (1:200, BD biosciences) for 30 min at 4°C. To detect the proteins involved in glycolysis, we used the following antibodies (HK1 clone C35C4, GLUT1 clone D3J3A and HIF-1α D1S7W - Cell Signaling - all at 1:500), followed by incubation with anti-Rabbit secondary Ab (PE anti-IgG H+L 1:1000, Invitrogen). The cells were acquired by LSRII (BD Biosciences) at the VUMC Flow Cytometry Shared Resource core (FCSR). Data were analyzed with FlowJo Version 10.

De Melo P., Rocio Pineros Alvarez A., Ye X., Blackman A., Alves-Filho J.C., Medeiros A.I., Rathmell J., Pua H, & Serezani C.H. (2021). Macrophage-derived microRNA-21 drives overwhelming glycolytic and inflammatory response during sepsis via repression of the PGE2/IL-10 axis.. Journal of immunology (Baltimore, Md. : 1950), 207(3), 902-912.

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Cytometric Analysis of T Cell Subsets

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Single-cell suspensions were prepared from lymph nodes, spleen, and lungs, as described (47 , 55 (link)). Unspecific staining was blocked with unconjugated anti-FcγRII/III antibody (anti-CD16/CD32, clone 2.4G2; BD Biosciences). Cells were stained with the following antibodies for multi-color cytofluorometric analyses: ECD-conjugated anti-CD8α (clone 53-6.7, Beckman Coulter), PE-conjugated anti-TCR Vβ5.1/5.2 (clone MR9-4, BD Biosciences), and APC-conjugated anti-TCR Vα2 (clone B20.1, BD Biosciences). Peptide/epitope-specific CD8⁺ T cells were identified with APC-conjugated MHC-I dextramer H-2Kb/SIINFEKL (Immudex, Copenhagen, Denmark). For the analyses, a “live gate” was routinely set on leukocytes in the forward scatter (FSC) versus sideward scatter (SSC) plot. All cytofluorometric analyses were performed with flow cytometer FC500 and CXP analysis software (Beckman Coulter).

Gergely K.M., Podlech J., Becker S., Freitag K., Krauter S., Büscher N., Holtappels R., Plachter B., Reddehase M.J, & Lemmermann N.A. (2021). Therapeutic Vaccination of Hematopoietic Cell Transplantation Recipients Improves Protective CD8 T-Cell Immunotherapy of Cytomegalovirus Infection. Frontiers in Immunology, 12, 694588.

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Multi-Color Flow Cytometry of Lung CD8+ T Cells

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Single-cell suspensions were prepared from lung tissue as described (22 (link), 48 (link)). Unspecific staining was blocked with unconjugated anti-FcγRII/III antibody (anti-CD16/CD32; clone 2.4G2, BD Pharmingen, Heidelberg, Germany). Cells were specifically stained with the following antibodies for multi-color cytofluorometric analyses: ECD-conjugated anti-CD8α (clone 53-6.7; Beckman Coulter, Krefeld, Germany), FITC-conjugated anti-KLRG1 (clone 2F1; eBioscience, Frankfurt), PE-Cy5-conjugated anti-CD127 (clone A7R34; eBioscience, Frankfurt), and PE-Cy7-conjugated anti-CD62L (clone MEL-14; Beckman Coulter). Phenotypic characterization of peptide-specific CD8⁺ T cells was performed using PE-conjugated dextramers H-2Ld/YPHFMPTNL (IE1), H-2Dd/AGPPRYSRI (m164), and H-2Kd/TYWPVVSDI (M105) (22 (link), 31 (link)). H-2Kb/SIINFEKL served as the control for excluding unspecific staining (Immudex, Copenhagen, Denmark). For the analyses, a “live gate” was routinely set on leukocytes in the forward scatter (FSC) versus sideward scatter (SSC) plot. All cytofluorometric analyses were performed with flow cytometer FC500 and CXP analysis software (Beckman Coulter).

Griessl M., Renzaho A., Freitag K., Seckert C.K., Reddehase M.J, & Lemmermann N.A. (2021). Stochastic Episodes of Latent Cytomegalovirus Transcription Drive CD8 T-Cell “Memory Inflation” and Avoid Immune Evasion. Frontiers in Immunology, 12, 668885.

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Isolation and Analysis of Murine Immune Cells

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Following preincubation with anti‐CD16/CD32 (clone 2.4G2 1:200, BD), BM cells were incubated with the following antibodies for 30 min at 4°C: CD11b (Clone M1/70 1:200, FITC, eBioscience, Sand Diego, CA); Ly6G (Clone 1A8 1:200, PerCP‐Cy5.5, BioLegend, San Diego, CA); Siglec‐F (Clone E50‐2440 1:200, APC‐Cy7, BD); CD3 (Clone 17A2 1:200, PE, BioLegend, San Diego, CA) and CD4 (Clone RM4.5 1:400, PE‐Cy7, BD). Then, cells were washed, resuspended in FACS buffer and stained with DAPI. Eosinophils, neutrophils, and CD4 T lymphocytes were sorted using a FACSAria III sorter (BD) in TRIzol LS reagent (Invitrogen), and RNA was isolated using the 5PRIME Phase Lock gel columns (Quantabio, Beverly, MA) and the PicoPure RNA Isolation Kit (Thermo Fisher Scientific). Reverse transcription to cDNA and qPCR were performed as described above.

Piñeiro‐Hermida S., Martínez P, & Blasco M.A. (2021). Short and dysfunctional telomeres protect from allergen‐induced airway inflammation. Aging Cell, 20(5), e13352.

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Peptide-Stimulated Splenocyte Cytokine Analysis

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Splenocytes were prepared as indicated above. Stimulation assays were performed using 1 × 10⁶ live splenocytes per well in 96-well U-bottom plates. Pools of overlapping peptides spanning the entire coding sequences of brachyury, CEA and MUC1 were synthesized as 15-mers with 11-amino acid overlaps (JPT GmbH) and lyophilized peptide pools were dissolved in Dimethyl sulfoxide (DMSO). Similarly constructed peptide pools corresponding to SIV-Vif and SIV-Nef served as off-target controls. Splenocytes in R10 media (RPMI 1640, 10% fetal bovine serum, and antibiotics) were stimulated by the addition of peptide pools at 2 μg/mL/peptide for 6 h at 37°C and 5% CO₂, with protein transport inhibitor (GolgiStop, BD) added 2 hours into the incubation. Stimulated splenocytes were then stained for lymphocyte surface markers CD8α and CD4, fixed, permeabilized, and then stained for the intracellular accumulation of IFN-γ and TNF-α. Antibodies against mouse CD8α (clone 53–6.7), CD4 (clone RM4–5), IFN-γ (clone XMG1.2), and TNF-α (clone MP6-XT22) were purchased from BD and staining was performed in the presence of anti-CD16/CD32 (clone 2.4G2). Flow cytometry was performed using an Accuri C6 Flow Cytometer (BD) and analyzed in BD Accuri C6 Software.

Gabitzsch E.S., Tsang K.Y., Palena C., David J.M., Fantini M., Kwilas A., Rice A.E., Latchman Y., Hodge J.W., Gulley J.L., Madan R.A., Heery C.R., Balint JP J.r., Jones F.R, & Schlom J. (2015). The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic. Oncotarget, 6(31), 31344-31359.

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