Cd16 cd32

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD16/CD32 is a laboratory equipment product that measures the expression of the Fc gamma receptors CD16 and CD32 on the surface of cells. It is used to analyze immune cell populations and their activation states.

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CD16/CD32 antibody (13 citations) Anti-mouse CD16/CD32 Fc block (12 citations) Rat anti-mouse CD16/CD32 (8 citations) CD16/CD32 (clone 93, (7 citations) CD16/CD32 (93) (6 citations) Anti-mouse CD16/CD32 monoclonal antibody (5 citations) Fc blocker (CD16/CD32, (3 citations) APC anti-mouse CD16/CD32 (2 citations) Anti-CD16/CD32 antibodies (clone 93; (2 citations) Fc-blocked (anti-CD16/CD32; (2 citations)

23 protocols using cd16 cd32

Macrophage Characterization and Polarization

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Single-cell suspensions of NR8383 cells and BMDMs were prepared as previously described. BMDMs were incubated first with CD16/CD32 (Thermo Fisher Scientific) and then with FITC-CD68 (MA5-28262, Thermo Fisher Scientific) and PERCPEF710-CD11b/c (46-0110-82, Thermo Fisher Scientific). The percentage of double-positive cells indicates the purity of macrophages. To identify macrophage polarization, cells were first incubated with CD16/CD32, followed by PE-CD86 (12-0860-83, Thermo Fisher Scientific) and CD163 (PA5-78961, Thermo Fisher Scientific). Finally, the cells were incubated in the dark for 1 h with APC (A-10931, Thermo Fisher Scientific). Apoptosis was determined by an Annexin V-FITC/PI double staining cell apoptosis detection kit (Sevenbio, Beijing, China) following the manufacturer’s instructions. Each sample was analyzed by flow cytometry and FlowJo v.10 software.

Tang D.S., Cao F., Yan C.S., Cui J.T., Guo X.Y., Cheng L., Li L., Li Y.L., Ma J.M., Fang K., Gao L., Ren N.S., Sun B., Wang G, & Ji L. (2022). Acinar Cell-Derived Extracellular Vesicle MiRNA-183-5p Aggravates Acute Pancreatitis by Promoting M1 Macrophage Polarization Through Downregulation of FoxO1. Frontiers in Immunology, 13, 869207.

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Multicolor Flow Cytometry Analysis

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The blood, spleen, and DLN were harvested on day 7 after treatment. Single-cell suspensions were prepared, and red blood cells were lysed using ACK Lysis Buffer. Antibodies targeting CD4 (GK1.5), CD8 (53–6.7), CD3 (145-2C11), Gr1 (RB6-8C5), CD11b (M1/70), CD45 (30-F11), CD62L (MEL-14), CD44 (IM7), PD-L1 (MIH5), and CD16/CD32 were purchased from eBioscience (Waltham, MA, USA). Antibodies targeting F4/80 (BM8), CD11c (N418), and Ki-67(SOIA15) were purchased from Biolegend (San Diego, CA, USA). Intracellular staining was performed using a fixation/permeabilization kit (eBioscience). The following gating strategies were used for flow cytometry: percentages of CD4⁺ and CD8⁺ after gating on live CD45⁺ CD3⁺ cells. The granulocytic MDSC population is CD11b⁺ and Gr1⁺. The Gr1− portion of the CD11b⁺ population was further subdivided by F4/80 staining. Gr1⁻ CD11b⁺ F4/80⁺ cells are macrophages. The DC population is CD11c⁺. Flow-cytometric analysis was performed using a FACSVerse (Becton Dickinson), and data were analyzed using FlowJo software.

Lin X., Zeng T., Xiong J., Zhang Q., Jiang P., Li X., Lin S., Xu Q., Weng H., Lai H., Gong H., Lin J., Cheng N., Tian X., Xu Y., Fang S., Jin R., Chen Z., Yang J., Morton L., Yueh B, & Lin J. (2018). Combined α-programmed death-1 monoclonal antibody blockade and fractionated radiation therapy reduces tumor growth in mouse EL4 lymphoma. Cancer Biology & Therapy, 20(5), 666-679.

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Quantification of Peritoneal Immune Cells

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Zymosan (0.1 mg) was injected i.p. into lipin-1^mKO and littermate control mice and allowed to incubate for 6 d. Mice were then sacrificed, and the peritoneal lavage was collected in FACS wash buffer (1% BSA, 1 mM EDTA, and 0.1% sodium azide in PBS). A total of 500,000 isolated peritoneal cells were blocked with CD16/CD32 (1:200) (14-0161-86; eBioscience) for 20 min. Cells were incubated with PECy7-conjugated CD11b (1:4000) (25-0112-81, clone M1/70; eBioscience), AF700-conjugated anti-CD45.2 (1:2000) (109821, clone 104; BioLegend), FITC-conjugated anti-Ly6G (1:800) (551460, clone 1A8; BD Biosciences), PECy5-conjugated anti-F4/80 (1:400) (15-4801-80, clone BM8; Invitrogen), and PE-conjugated anti-MerTK Ab (1:500) (151506; BioLegend) for 30 min in the dark. Cells were spun at 400 × g for 5 min and resuspended in FACS fix (1% paraformaldehyde in FACS wash buffer). Appropriate F Minus One Controls were used to identify positive staining. Compensation controls (Comp Bead, 01-2222-42; Invitrogen) were used to exclude spectral overlap. Flow cytometry was performed using Quanteon flow cytometer (Acea Biosciences). Data analysis was performed using FCS express (Denovo Software) and NovoExpress (Acea Biosciences).

Schilke R.M., Blackburn C.M., Rao S., Krzywanski D.M., Finck B.N, & Woolard M.D. (2020). Macrophage-Associated Lipin-1 Promotes β-Oxidation in Response to Proresolving Stimuli. ImmunoHorizons, 4(10), 659-669.

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Hematopoietic Lineage Analysis in Knockout Mice

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For analysis of hematopoietic lineage markers in the BMs of mice with specific knockout of Wls or Six1 in hematopoietic cells, we follow protocols described previously [30 (link)]. Characterization of MLL-AF9 AML and isolation of the enriched LIC population from BMs of AML mice have been described previously [28 (link)]. Fluorescein-labeled antibodies used in the study include: Biotin Mouse Lineage Depletion Cocktail (BD Biosciences); Streptavidin-PerCP-Cy5.5, Sca1-FITC, cKit-APC-Cy7, cKit-PE, FcγR-PE-Cy7, CD34-PacificBlue, FLT3-PE, IL7R-APC, Mac1-APC, B220-PE, CD3-APC, Gr1-PE, Mac1-APC, CD16/CD32, all antibodies are anti-mouse and were purchased from eBiosciences.

Zhang L.S., Kang X., Lu J., Zhang Y., Wu X., Wu G., Zheng J., Tuladhar R., Shi H., Wang Q., Morlock L., Yao H., Huang L.J., Maire P., Kim J., Williams N., Xu J., Chen C., Zhang C.C, & Lum L. (2018). Installation of a cancer promoting WNT/SIX1 signaling axis by the oncofusion protein MLL-AF9. EBioMedicine, 39, 145-158.

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Cellular Phenotyping via Flow Cytometry

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Anti-mouse B220, CD11b, CD11c, CD19, CD31, CD45, CD16/CD32, F4/80, and MHC class II (MHCII) mAbs were purchased from eBioscience (San Diego, CA, USA). Appropriate isotype-matched control antibodies were used to determine nonspecific staining. ATII-like cells (ATII-LCs) (CD11b^-/CD11c^-/CD16/32^-/CD19^-/CD31^-/CD45^-/F4/80^-MHCII⁺), macrophages (CD11b⁺/CD11c^-), DCs (CD11b^-/CD11c⁺), and B cells (B220⁺/CD19⁺) were stained using standard procedures and analysed by flow cytometry.

Akgün J., Schabussova I., Schwarzer M., Kozakova H., Kundi M, & Wiedermann U. (2015). The Role of Alveolar Epithelial Type II-Like Cells in Uptake of Structurally Different Antigens and in Polarisation of Local Immune Responses. PLoS ONE, 10(4), e0124777.

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Lymphocyte Immunophenotyping at Weaning and Adolescence

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After euthanasia, cell suspensions of spleen (at weaning and 5 days) and mesenteric lymphoid nods (only at weaning) were obtained by mechanically extrusion through a 40-μm nylon cell strainer (BD, Switzerland). Cells were washed through the strainer using 1 ml of Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, France) supplemented with 10% foetal bovine serum (FBS, Gibco). Erythrocytes were lysed by incubation with the red blood cell lysing buffer Hybri-Max (Sigma-Aldrich) according to manufacturer instructions. For each sample, aliquots of 10⁶ cells were transferred to two 96-well plates (Grenier, France). Following standard protocols as previously described [9 (link), 30 (link)], cells were stained with one of the next: (i) anti-CD4-FITC, anti-CD3e-percp and anti-T-bet-PE; (ii) anti-CD4-FITC, anti-CD3e-percp and anti-Gata3-PE; (iii) anti-CD4-FITC, anti-CD3e-percp and anti-rorγ-PE; or (iv) anti-CD4-FITC, anti-CD3e-percp and anti-Foxp3-PE (all from eBioscience, France). All stainings were performed in the presence of CD16/CD32 (eBioscience). Samples were subsequently analysed using an Accuri C6 cytometer (BD). The data obtained from the cytofluorimetric analysis were processed using CFlowSampler software (BD).

Barone M., Ramayo-Caldas Y., Estellé J., Tambosco K., Chadi S., Maillard F., Gallopin M., Planchais J., Chain F., Kropp C., Rios-Covian D., Sokol H., Brigidi P., Langella P, & Martín R. (2023). Gut barrier-microbiota imbalances in early life lead to higher sensitivity to inflammation in a murine model of C-section delivery. Microbiome, 11, 140.

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Post-Stroke Immune Cell Isolation

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Twenty-four hours after stroke, mice were euthanized, and single-cell suspensions were made of the ischemic (ipsilesional) hemispheres as previously described (68 (link)). Cells were incubated with CD45 APC-Cy7 (103116, BioLegend), CD11b PE-Cy7 (25-0112-82, eBioscience), Ly6G BV510 (127633, BioLegend), and CD16/CD32 (14-0161-82, Fc block, eBioscience) for 30 minutes at room temperature in PBS plus 5% FBS. Thirty minutes later, cells were washed, fixed, and run on a Beckman Coulter Cytoflex located in the Utah Flow Cytometry Core.

Denorme F., Portier I., Rustad J.L., Cody M.J., de Araujo C.V., Hoki C., Alexander M.D., Grandhi R., Dyer M.R., Neal M.D., Majersik J.J., Yost C.C, & Campbell R.A. (2022). Neutrophil extracellular traps regulate ischemic stroke brain injury. The Journal of Clinical Investigation, 132(10), e154225.

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Isolation and Sorting of Astrocytes and Microglia

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The obtained cell suspension was incubated with CD16/CD32 (1:100, eBioscience) to block Fc gamma receptors and prevent monocyte isolation. After washing, cells were resuspended and incubated with anti‐GLT‐1 (1:100, 0.6 mg/mL (Orre et al., 2014b) in staining medium (HBSS, PAA, 1.3% d‐(+)‐glucose solution, Sigma, Cat.nr. G8769; 1.5% HEPES, PAA, 311‐001, 0.1 mM EDTA) at 4°C for 30 min. After washing, cells were incubated with the secondary antibody anti‐rabbit‐Alexa488 (1:200) and the conjugated antibodies CD45‐PeCy7 (1:200 eBioscience) and CD11B‐PE (1:150, eBioscience) for 30 min at 4°C. Cells were washed with staining medium and 4′,6‐diamidino‐2‐fenylindool (DAPI) staining was used to sort living cells. Using an MoFlo XDP sorter (Beckman Coulter), GLT⁺ astrocytes were sorted based on a GLT⁺/CD11B⁻ expression and microglia were sorted on CD11B⁺/CD45⁺ expression. The sorted cells were subjected to RNA isolation.

Jansen A.H., van Hal M., op den Kelder I.C., Meier R.T., de Ruiter A., Schut M.H., Smith D.L., Grit C., Brouwer N., Kamphuis W., Boddeke H.W., den Dunnen W.F., van Roon W.M., Bates G.P., Hol E.M, & Reits E.A. (2016). Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell‐type‐specific. Glia, 65(1), 50-61.

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Immunohistochemical Staining of Dectin-1 and CD16/CD32 in Mouse Brain Sections

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Brain sections (14 μm) from snap-frozen mouse brains were acetone-fixed for 10 min, pre-incubated in Peroxidase Blocking Reagent (DAKO, K4009) for 15 min, and blocked in 5% fetal bovine serum for 30 min (RT). Sections were incubated with primary antibodies at RT for 2 h: Dectin-1 (1:100, AbDSerotec, MCA2289) or CD16/CD32 (1:100, eBioscience, 14-0161-82). Next, sections were incubated with unconjugated rabbit anti-rat IgG (Vector Laboratories, AI-4001) at RT for 1 h. Then sections were incubated with labeled polymer-HRP anti-rabbit (DAKO, K4009) at RT for 30 min. The complex was visualized after 10 min of incubation with 3-amino-9-ethylcarbazole (AEC) substrate-chromogen solution (DAKO, K4009) and counterstained with hematoxylin. The visualization and counterstaining were both done at RT.

Yin Z., Raj D.D., Schaafsma W., van der Heijden R.A., Kooistra S.M., Reijne A.C., Zhang X., Moser J., Brouwer N., Heeringa P., Yi C.X., van Dijk G., Laman J.D., Boddeke E.W, & Eggen B.J. (2018). Low-Fat Diet With Caloric Restriction Reduces White Matter Microglia Activation During Aging. Frontiers in Molecular Neuroscience, 11, 65.

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Isolation of Liver Non-Parenchymal Cells

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Mice were anesthetized, livers perfused with sterile PBS, and non‐parenchymal liver cells isolated as previously described (with slight modifications).24 Briefly, after successful perfusion, whole livers were excised and minced, incubated with 0.2 mg/mL collagenase IV buffer at 37°C for 30 minutes with shaking, double‐passed through 70 μm nylon mesh and centrifuged twice for 3 minutes at 40 g to pellet hepatocytes. Supernatants containing non‐parenchymal cells were purified in the Percoll gradient (Sigma‐Aldrich). Isolated cells were counted in hemocytometer (Bürker‐Türk chamber) and labelled with fluorescent‐conjugated antibodies: CD45‐APC (eBioscience), CD3‐APCeF780 (eBioscience), B220‐PECy7 (eBioscience), NK1.1‐PE (eBioscience), CD11b‐PECy7 (eBioscience), F4/80‐APCeF780 (eBioscience), Ly6G‐PE (BD Pharmingen) and CD95‐AF488 (eBioscience). CD16/CD32 (eBioscience) was used for blocking of Fc receptors, while dead cells were excluded based on 7‐amino‐actinomycin D (7‐AAD; BioLegend) binding. Upon labelling, cells were acquired and analysed using the Attune instrument (Thermo Fisher Scientific) and FlowJo software (FlowJo). The gating strategy is shown in Supporting Information (Figure S1).

Markotic A., Flegar D., Grcevic D., Sucur A., Lalic H., Turcic P., Kovacic N., Lukac N., Pravdic D., Vukojevic K., Cavar I, & Kelava T. (2020). LPS‐induced inflammation desensitizes hepatocytes to Fas‐induced apoptosis through Stat3 activation—The effect can be reversed by ruxolitinib. Journal of Cellular and Molecular Medicine, 24(5), 2981-2992.

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