Anti cd16 32

Manufactured by BioXCell

Sourced in United States

Anti-CD16/32 is a laboratory reagent that can be used to block the CD16 and CD32 receptors on cells. It is often used in flow cytometry and cell-based assays to prevent non-specific binding of antibodies or other ligands to these Fc receptors.

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Lab products found in correlation

Easysep biotin positive selection kit, by STEMCELL (2 mentions) Anti f4 80 alexafluor647, by BioLegend (1 mentions) Cy 3 affinipure donkey anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions) Lsrfortessa sorp cytometer, by BD (1 mentions) Bv421 conjugated anti cd64, by BioLegend (1 mentions) Zombie aqua fixable dye, by BioLegend (1 mentions) Pen strep glut, by Thermo Fisher Scientific (1 mentions) Ficoll paque premium 1, by GE Healthcare (1 mentions) Fetal calf serum (fcs), by Benchmark Scientific (1 mentions) Facsaria 2, by BD (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Lysis buffer, by Thermo Fisher Scientific (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Facsaria fusion, by BD (1 mentions) β mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Cd11b af594 clone m1 70, by BioLegend (1 mentions) Bovine serum albumin (bsa), by BioXCell (1 mentions) Foxp3 fix perm kit, by BD (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Vectashield, by Vector Laboratories (1 mentions)

Spelling variants of this product

Anti-mouse CD16/32 (24G2; (6 citations) Anti-CD16/32 (clone 2.4G2, (5 citations) Anti-CD16/32 antibody (2.4G2 clone; (4 citations) Rat anti-mouse CD16/32 (clone 2.4G2, (3 citations) CD16/32 (2 citations) Anti-mouse CD16/32 (2 citations) Anti-CD16/32 antibody (2.4G2, (2 citations) Anti-CD16/32 Fc-block (2 citations) Fc Block (anti-CD16/32 clone 2.4G2; (2 citations) Anti-CD16/32 mAb (2.4G2, (2 citations)

9 protocols using anti cd16 32

Multiparametric flow cytometry analysis

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Single cell suspensions were incubated with anti-CD16/32 (BioXcell) for 15 min at 4 °C, followed by staining with fluorescently conjugated antibodies for 30–45 min at 4 °C.
The following cell populations were identified based on cell marker expression: neutrophils (CD45⁺CD11b⁺Ly6C⁻Ly6G⁺), monocytes (CD45⁺ CD11b⁺Ly-6G⁻Ly-6C^high), CD11b⁺ macrophage (CD45⁺CD11b⁺Ly6G⁻Ly6C⁻F4/80⁺), T cells (CD45⁺CD3⁺CD4⁺ or CD8⁺), B cells (CD45⁺B220⁺CD19⁺), NK cells (CD45⁺CD3⁻NK1.1⁺), DC1 (CD45⁺CD3⁻CD19⁻NK1.1⁻, MHCII⁺CD11c⁺ CD64⁻CD11b⁻CD103⁺ (tumour) XCR1⁺ (lymphoid tissue)), DC2 (CD45⁺ CD3⁻CD19⁻NK1.1⁻MHCII⁺CD11c⁺CD64⁻CD11b⁺CD103/XCR1⁻), migDC DC1 (CD45⁺CD3⁻CD19⁻NK1.1⁻MHCII⁺CD11c⁺CD64⁻CD11b⁺CD103⁺).
The following antibodies (Biolegend and BD) were used: CD45 (30-F11), CD4 (RM4-5), CD8a (53-6.7), B220 (RA3-6B2), NK1.1 (PK136), CD11b (M1/70), CD11c (N418), CD103 (P84), XCR1 (ZET), CD64 (X54-5/7.1), CD86 (GL-1), PDL1 (10F.9G2), CD27 (LG.3A10), IL-7Ra (A7R34), CXCR5 (L138D7), CX3CR1 (SA011F11), CD5 (53-7.3), CD24 (M1/69), PDL2 (122), Lag3 (C9B7W), TIGIT (GIGD7), TIM-3 (5D12), Ly6C (HK1.4), Ly6G (1A8), TCRb (H57-597), F4/80 (BM8), Ki67 (16A8), T-bet (4B10) and TCF1 (C63D9).

Dixon K.O., Tabaka M., Schramm M.A., Xiao S., Tang R., Dionne D., Anderson A.C., Rozenblatt-Rosen O., Regev A, & Kuchroo V.K. (2021). TIM-3 restrains anti-tumour immunity by regulating inflammasome activation. Nature, 595(7865), 101-106.

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Flow Cytometric Immunophenotyping of Cells

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Single-cell suspension was stained with anti-CD16/32 (BioXcell, 1 mg ml⁻¹, 1:100) and conjugated antibodies against any combination of the following surface antigens: CD4 (RM4.5, 1:400), CD8 (53-6.7, 1:400), CD45 (30-F11, 1:400), TCRβ (H57-597, 1:200) and PD-1 (29F.1A12, 1:200). For examination of cytokines and cellular proliferation, conjugated antibodies against any combination of the following intracellular antigens were used: TNF (MP6-X122, 1:200), IFNγ (XMG1.2, 1:200) and Ki-67(16A8, 1:1,000).

Karagiannis F., Peukert K., Surace L., Michla M., Nikolka F., Fox M., Weiss P., Feuerborn C., Maier P., Schulz S., Al B., Seeliger B., Welte T., David S., Grondman I., de Nooijer A.H., Pickkers P., Kleiner J.L., Berger M.M., Brenner T., Putensen C., Kato H., Garbi N., Netea M.G., Hiller K., Placek K., Bode C, & Wilhelm C. (2022). Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19. Nature, 609(7928), 801-807.

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Immunostaining of Testicular Frozen Sections

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For immunostaining using frozen sections, testes were embedded in optimum cutting temperature compound (O.C.T.) and frozen in liquid nitrogen. Serial sections were cut at 5 μM thickness onto glass slides and stored at −80°C until use. Slides were blocked 1 h at room temperature in PBS/5% BSA/1% mouse serum/1% anti-CD16/32 (BioXcell; clone 2.4G2). Primary and secondary antibodies were added, followed by staining with DAPI. The following antibodies were used throughout this study: anti-F4/80-AlexaFluor647 (BioLegend; clone BM8), CD3ϵ (Santa Cruz Biotechnology; clone M-20), SOX9 (abcam; clone EPR14335-78), Biotin-SP-conjugated Affinipure Donkey Anti-Goat IgG (H+L) (Jackson immunoresearch; code:705-065-147), Cy™3 conjugated Sreptavidin (Jackson immunoresearch; code:016-160-084), Cy™3 AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson immunoresearch; code: 711-165-152).

Jing Y., Cao M., Zhang B., Long X, & Wang X. (2021). cDC1 Dependent Accumulation of Memory T Cells Is Required for Chronic Autoimmune Inflammation in Murine Testis. Frontiers in Immunology, 12, 651860.

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Ex vivo CD45+ Cell Coupling Assay

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Ex vivo coupling assays were performed as described previously (Broz et al., 2014 (link)). Briefly, single cells suspensions were enriched for CD45⁺ cells using EasySep biotin positive selection kit (Stemcell Technologies). Enriched cells were stained with with Zombie NIR Fixable live/dead dye (Biolegend) for 20 min at 4°C, followed by staining for 30 min at 4°C with directly conjugated antibodies diluted in FACS buffer containing anti-CD16/32 (BioXCell) to block non-specific binding. Cells were washed again and co-cultured with VPD-labeled previously activated OT-I CD8⁺ T cells for 1 hour at 37°C. Cells were lightly fixed in 2% PFA followed by read-out on a BD LSR Fortessa SORP cytometer.

Kersten K., Hu K.H., Combes A.J., Samad B., Harwin T., Ray A., Rao A.A., Cai E., Marchuk K., Artichoker J., Courau T., Shi Q., Belk J., Satpathy A.T, & Krummel M.F. (2022). Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells in cancer. Cancer cell, 40(6), 624-638.e9.

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Bone Marrow-Derived Macrophage Viability

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BMDMs were stained for viability using the Zombie Aqua Fixable dye (BioLegend), followed by Fc blocking using 20 µg/mL anti-CD16/32 (BioxCell, clone 2.4G2) for 20 min. Cells were stained with BV421-conjugated anti-CD64 (BioLegend, clone X54–5/7.1) and acquired on a BD LSRII. Data were analyzed using FlowJo v10 (BD Biosciences).

Thomas S.A., Yong H.M., Rule A.M., Gour N, & Lajoie S. (2024). Air pollution drives macrophage senescence through a phagolysosome-15-lipoxygenase pathway. bioRxiv.

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Isolation and Sorting of Tumor-Infiltrating Cells

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Single cell suspensions from tumors were prepared as described above. For T cell isolations, single cell suspensions were enriched for mononuclear cells using Ficoll-Paque Premium 1.084 (GE Healthcare). For isolation of myeloid cells, single cell tumor suspensions were enriched for CD45+ cells using EasySep biotin positive selection kit (Stemcell Technologies). Enriched cells were stained for 30 min at 4°C with directly conjugated antibodies diluted in FACS buffer containing anti-CD16/32 (BioXCell) to block non-specific binding. Cells were washed again with FACS buffer and filtered over a 70μm mesh. Immediately prior to sorting, DAPI was added to exclude dead cells. Cells were sorted on a BD FACSAria Fusion and BD FACSAria2. Sorted T cells were collected directly in lysis buffer (Invitrogen) for RNA sequencing or in RPMI (GIBCO), 10% FCS (Benchmark), Pen/Strep/Glut (Invitrogen) and 50μM β-mercaptoethanol (GIBCO) at 4°C for further use ex vivo. Sorted myeloid cells were collected in DMEM (GIBCO), 10% FCS (Benchmark), Pen/Strep/Glut (Invitrogen) at 4°C for further use ex vivo.

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Hypoxia Mapping in Murine Tissues

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Mice were injected with pimonidazole hydrochloride in PBS (80mg/kg; Hypoxyprobe) intraperitoneally 1.5 hours prior to sacrifice. Tissues were dissected and processed as described above. For flow cytometry studies, pimonidazole was visualized using anti-pimonidazole antibodies (Pacific Blue Mab-1 clone 4.3.11.3; Hypoxyprobe) after cells were fixed for 20 min at 4°C using the FOXP3 Fix/Perm kit (BD Biosciences), and washed in permeabilization buffer. For imaging studies, dissected tumors were embedded in OCT and sectioned into 10μm cryosections. Cryosections were stored at −80°C until further use. For immunostaining, sections were fixed in 4% PFA (Electron Microscopy Sciences) for 20 minutes at RT, followed by a rinse in PBS containing 1% BSA (Sigma). Sections were blocked in 1% BSA in PBS containing anti-CD16/32 (BioXCell) for 1 hour at RT, and washed. Sections were stained with CD11b-AF594 (clone M1/70;BioLegend), CD31-AF647 (clone 390;BioLegend) and Pacific Blue Mab-1 (clone 4.3.11.3;Hypoxyprobe) for 1 hour at RT, followed by a wash and mounted using Vectashield (Vector Laboratories) and sealed with nail polish. Images were acquired on a Leica SP8 confocal microscope. Data analysis was performed using Imaris (Bitplane).

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Purification and Labeling of OTI CD8+ T Cells

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OTI cells were purified from the spleens of CD45.1+ OTI mice by incubating single cell suspensions with anti-CD16/32 (BioXcell) and anti–CD8 magnetic beads (Miltenyi Biotec), for 45 minutes at 4°C. After washing, CD8⁺ cells were enriched by magnetic separation using LS–columns (Miltenyi Biotec) and stained with cell trace violet (CTV, Thermo–Fisher) according to instructions. Cells were adjusted to 6 x 10⁵ cells/mL in sterile PBS and 50 μL of the cell suspension were transferred intraperitoneally. For DC enrichment, cell suspensions from dissociated lungs were incubated with anti–CD11c magnetic beads followed by magnetic separation using LS columns (Miltenyi Biotec). After enrichment, DCs were purified by first gating on Lin⁻CD64⁻MHCII⁺CD11c⁺ cells and sorted based on expression of CD103, CD11b, SIRPα. Post–sorting purity was >95%. All sorting was performed using a FACSAriaII provided by the Flow Cytometry and Single Cell services Core at UAB.

, & Nagylaki T. (1991). Error bounds for the fundamental and secondary theorems of natural selection.. Proceedings of the National Academy of Sciences of the United States of America, 88(6), 2402-2406.

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Murine Immune Cell Profiling for Malaria

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Male BALB/c mice (6–8 weeks old) used for this study were maintained under pathogen-free condition at institutional animal house facility. Anti-mouse antibodies Alexa Fluor 700-CD8 (Clone 53-6.7), APC-cy7-CD19 (Clone 1D3), APC-cy7-CD45R/B220 (Clone RA3-6B2), were procured from BD Biosciences (San Jose, CA, USA), FITC-CD4 (clone GK 1.5), FITC CD278 (7E.17G9), APC-CD278 (Clone-C398.4A), PerCP-Cy 5.5-CD4 (clone RM4-5), PE-Cy7-IFN-γ (clone XMG 1.2), PE-T-bet (clone eBio 4B10), Alexa Fluor 700-CD8 (53-6.7) were from eBioscience (San Diego, CA, USA), Brilliant violet 605 TCR Vb (clone H57-597), APC-CD278 (clone C398.4A), Anti-CD49b (clone DX5) were from BioLegend (San Diego, CA, USA). Purified antibodies Anti-mouse CD278 ICOS (clone 7E.17G9), Anti-Rat IgG2b isotype control, and anti-CD16/32 were procured from BioXcell (West Lebanon, USA). Dynabeads untouched mouse CD4⁺ T cell isolation kit was procured from Life Technologies AS (Oslo, Norway) and CD8⁺ T cell negative selection kit was from Stem cell technologies (Vancouver, BC, Canada). Phorbol 12-myristate 13-acetate (PMA), Ionomycin, Brefeldin A was procured from Sigma-Aldrich (St. Louis, MO, USA). Malarial parasite P. berghei ANKA (MRA-671, MR4, ATCC, Manassas, VA, USA) was obtained from MR4 repository, ATCC, Manassas, VA, USA.

Jogdand G.M., Sengupta S., Bhattacharya G., Singh S.K., Barik P.K, & Devadas S. (2018). Inducible Costimulator Expressing T Cells Promote Parasitic Growth During Blood Stage Plasmodium berghei ANKA Infection. Frontiers in Immunology, 9, 1041.

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