Rat anti mouse cd16 32

Manufactured by BD

The Rat anti-mouse CD16/32 is a laboratory reagent used to detect and characterize the expression of CD16 and CD32 receptors on mouse cells. It is a tool for immunological research and analysis.

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Anti-CD16/32 (78 citations) CD16/32 (49 citations) Anti-mouse CD16/32 (45 citations) Rat anti-mouse CD16/32 antibody (18 citations) Rat anti-CD16/32 (14 citations) Mouse anti (2 citations) Rat anti-mouse CD16/32 (2.4G2, (2 citations) Purified rat anti-mouse CD16/32 (2 citations) Rat-anti-mouse Abs to CD16/32 (2.4 G; (2 citations)

11 protocols using rat anti mouse cd16 32

Comprehensive Antibody Panel for Actin Cytoskeleton Analysis

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The following antibodies were used in this study: rabbit anti-myo1e has been previously described^{77 (link)}; myo1f (Santa Cruz, B-5, #376534); capping protein (Millipore, #AB6017); Arp3 (Millipore, clone 13C9, #MABT95); BSA (Sigma, clone 3H6, #SAB5300158); rat anti-mouse CD16/32 (BD, #553141); AffiniPure mouse anti-rat (Jackson Labs, 212-005-082); the following antibodies were purchased from Cell Signaling Technologies: pSyk (#2701), Syk (#13198), pAkt (#4060), Akt (#4691), pERK (#4370), ERK (#9102); fluorescent secondary antibodies against mouse or rabbit (Life Technologies). CARMIL1 antibody (WU-C101) was a gift from John Cooper, Washington University in St. Louis (St. Louis, MO).

Barger S.R., Reilly N.S., Shutova M.S., Li Q., Maiuri P., Heddleston J.M., Mooseker M.S., Flavell R.A., Svitkina T., Oakes P.W., Krendel M, & Gauthier N.C. (2019). Membrane-cytoskeletal crosstalk mediated by myosin-I regulates adhesion turnover during phagocytosis. Nature Communications, 10, 1249.

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Splenic Immune Cell Isolation Protocol

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Upon animal sacrifice, spleens were mechanically broken down with surgical scissors and ground through a 40 μm strainer with a plastic plunger of a 1 mL syringe into a 50 mL conical tube. The strainer was washed with 5 mLs of FACS Buffer (PBS with 3% w/v bovine serum albumin (BSA)). After centrifugation at 176 x g, cells were resuspended in 2 mLs Pharmlyse Buffer for 2 minutes in a 37 C water bath. After incubation, 40 mLs of FACS buffer were added to the samples. Cell viability was assessed using Trypan Blue. To minimize non-specific Fc receptor binding, rat anti-mouse CD16/32 (BD Pharmingen) was used as an Fc block. Splenic cells were stained with PE rat anti-mouse CD19 (BD Pharmingen), PE-Cy7 anti-mouse B220 (Invitrogen), FITC rat anti-mouse CD3, and APC anti-mouse CD4 (Invitrogen).

Macbeth J.C., Liu R., Alavi S, & Hsiao A. (2021). A dysbiotic gut microbiome suppresses antibody mediated-protection against Vibrio cholerae. iScience, 24(12), 103443.

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Immunofluorescence Analysis of Spinal Cord and DRG

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Spinal cord and DRG were post-fixed in 4% paraformaldehyde and cryoprotected in sucrose. Cryosections (10-μm) of lumbar DRG and of lumbar segments L2–L5 were stained. We used 1:500 rat antimouse CD16/32 (BD Bioscience) or 1:500 rabbit anti-Iba- 1 (Wako Pure Chemical Industries, Richmond, VA), 1:100 rabbit anti-GFP (GeneTex, San Antonio, TX), and 1:200 rat anti-CD45 (BD Bioscience), followed by alexafluor 488-conjugated streptavidin or alexafluor 488- or 594-conjugated secondary antibodies. Photographs were taken with a Zeiss Axio Observer microscope (Zeiss, Oberkochen, Germany).

Willemen H.L., Eijkelkamp N., Carbajal A.G., Wang H., Mack M., Zijlstra J., Heijnen C.J, & Kavelaars A. (2014). Monocytes/Macrophages Control Resolution of Transient Inflammatory Pain. The journal of pain : official journal of the American Pain Society, 15(5), 496-506.

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Murine Macrophage Profiling of L. braziliensis

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BMDM were exposed to L. braziliensis according to strategies #1 and #2 (Fig. 4) for 18 h in 5 ml polypropylene tubes. Cell samples were harvested for staining with fluorochrome-labeled antibodies specific to F4/80, CD11b, CD40 and CD86 (Filters used: PE, PE/Cy7, FITC and BV 421, respectively) according to established protocols. Briefly, cells were first blocked at 4 °C with rat anti-mouse CD16/32 (5 mg/ml; BD Pharmingen) for 10 min and then stained with anti-mouse antibodies for 30 min (1:200 dilution each molecule) at 4 °C. Cells were also stained with Fixable Viability Dye (eBioscience/ThermoFisher) to recognize dead cells for exclusion. Cells were finally fixed with 2% paraformaldehyde in PBS for 10 min, washed by centrifugation with washing buffer and kept at 4 °C in the dark until data acquisition. Fluorescence-minus-one (FMO) controls for CD40 and CD86 were included in all experiments. Data were acquired in a Fortessa flow cytometer (BD Biosciences, USA) and analyzed by using FlowJo software (Tree Star, Version 10.2). Macrophages were pre-gated by surface phenotype (F4/80 and CD11b positive cells).

Sharma R., Viana S.M., Ng D.K., Kolli B.K., Chang K.P, & de Oliveira C.I. (2020). Photodynamic inactivation of Leishmania braziliensis doubly sensitized with uroporphyrin and diamino-phthalocyanine activates effector functions of macrophages in vitro. Scientific Reports, 10, 17065.

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Flow Cytometric Analysis of Cell Surface and Intracellular Markers

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Cells were stained with Live/Dead fixable aqua dye (ThermoFisher Scientific) for 10 min at room temperature and were then washed with 1% FBS in PBS (Lonza). Nonspecific binding of antibodies was blocked by incubating cells with rat anti-mouse CD16/32 (BD Biosciences) at 4 °C for 10 min, followed by incubating with fluorochrome-conjugated antibodies recognising cell-surface markers for 30 min at 4 °C. Following by two wash steps, cells were fixed using either BD fix (BD Biosciences) for 10 min at 4 °C or Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher Scientific) for 60 min at 4 °C for intracellular staining. For intracellular staining, fixed and permeabilised cells were washed and stained with fluorochrome-conjugated antibodies against intracellular markers. Cell acquisition was performed using an LSRII (BD Biosciences) or LSRFortessa (BD Biosciences) flow cytometer. The data were analysed using FlowJo 10.5.3.

Park I., Goddard M.E., Cole J.E., Zanin N., Lyytikäinen L.P., Lehtimäki T., Andreakos E., Feldmann M., Udalova I., Drozdov I, & Monaco C. (2022). C-type lectin receptor CLEC4A2 promotes tissue adaptation of macrophages and protects against atherosclerosis. Nature Communications, 13, 215.

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Quantifying Tumor Cell Internalization of RG7787

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Animals were treated with RG7787-Alexa Fluor 647 or 488 when tumors reached an average of 120-140 mm13 . Animals were sacrificed at prescribed times, and tumors were excised. Digested tumor samples were prepared as previously reported13 . Samples were stained with 10 μg/mL rat anti-mouse CD16/32 (BD Pharmingen) in D-PBS 5% FBS 1 mM EDTA. Samples were stained with anti-CD71 R-PE (transferrin receptor; BioLegend) or anti-EGFR R-PE (BD Pharmingen) followed by staining with SytoxBlue Dead Cell Stain (Life Sciences). Data were collected using an LSRII (BD Biosciences). Gating and data analysis were done in FlowJo 10 software (Tree Star, Inc.). Cells were gated to exclude SytoxBlue stained cells and doublets, tumor cells were selected by gating for CD71 or EGFR positive cells (gating for representative control sample in Supporting Information Fig. 4). The amount of internalized RG7787-Alexa Fluor 647 was assessed by measuring the MFI of Alexa Fluor 647 in the tumor cell populations.

Mason-Osann E., Hollevoet K., Niederfellner G, & Pastan I. (2015). Quantification of recombinant immunotoxin delivery to solid tumors allows for direct comparison of in vivo and in vitro results. Scientific Reports, 5, 10832.

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Investigating the Regulation of Antigen Presentation by IL-10 in Dendritic Cells

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BMDCs (day 7) or splenic DCs were treated with 200 ng/mL IL-10 (BioLegend) or PBS for 16 h before being incubated with
100 |ig/mL DO Ovalbumin (ex. 505 nm, em. 515 nm; Molecular Probes) and 200 ng/mL IL-10 (BioLegend) or PBS for 20 m at
37°C or 0°C. Samples were then extensively washed with cold PBS + 2% FBS, stained in PBS + 2% FBS + 1 mM EDTA
for 20 m on ice, and DC populations and DO OVA were identified and quantified by flow cytometry using the MACSQuant (Miltenyi
Biotech) flow cytometer and analyzed by FlowJo software (Tree Star, Inc). The DQ-OVA was quantified using the FITC channel.
The following antibodies were used for staining DC subsets (from BioLegend unless specified): rat anti-mouse CD16/32 (BD
Biosciences), LIVE/DEAD Fixable Aqua (Invitrogen), Pacific Blue anti-mouse I-A/I-E (M5/114.15.2), PerCP/Cy5.5 anti-mouse/human
CD11 b (M1/70), PE/Cy7 anti-mouse TCRβ (H57–597), PE/Cy7 anti-mouse/human CD45R/B220 (RA3–6B2), APC
anti-mouse/rat XCR1 (ZET), APC/Fire 750 anti-mouse CD11 c (N418).

Liu D., Yin X., Olyha S.J., Nascimento M.S., Chen P., White T., Gowthaman U., Zhang T., Gertie J.A., Zhang B., Xu L., Yurieva M., Devine L., Williams A, & Eisenbarth S.C. (2019). IL-10-Dependent Crosstalk between Murine Marginal Zone B Cells, Macrophages, and CD8α+ Dendritic Cells Promotes Listeria monocytogenes Infection. Immunity, 51(1), 64-76.e7.

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Multiparametric Analysis of Immune Cells

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The ear dermis/ ear dLN/ lymph nodes/ spleen was removed from animals and single cell suspension was prepared. For surface staining, cells were blocked at 4°C with rat anti-mouse CD16/32 (5 μg/ml) from BD Pharmingen for 20 min. Cells were then stained with anti-mouse Ly6G, anti-mouse CD11b, anti-mouse CD3, anti-mouse CD4, anti-mouse CD44, anti-mouse CCR7, anti-mouse PD-1 (eBioscience) for 30 min (each with 1:200 dilution; at 4°C). The cells were then stained with Live/Dead fixable aqua (Invitrogen/Molecular Probes) to stain dead cells. Cells were washed twice with wash buffer and fixed with a Cytofix/Cytoperm kit (BD Bioscience) for 20 min at room temperature. Intracellular staining was performed with anti-mouse IFN-γ, anti-mouse IL-2, anti-mouse IL-10 (eBioscience) (each with 1:300 dilution; at 4°C). Cells were washed twice with permeabilization buffer and acquired on an LSR II (BD Biosciences) equipped with 407-, 488-, 532-, and 633-nm laser lines using FACS Diva 6.1.2 software. Data were analyzed with FlowJo software version 9.7.5 (Tree Star). For analysis, first doublets were removed using width parameter; dead cells were excluded based on staining with the Live/Dead aqua dye. Lymphocytes were identified according to their light-scattering properties. CD4 and CD8 T cells were identified as CD3⁺ lymphocytes uniquely expressing either CD4 or CD8.

Bhattacharya P., Dey R., Saxena A., Karmakar S., Ismail N., Gannavaram S., Dagur P.K., Satoskar M., Satoskar S., De Paoli S., Takeda K., McCoy JP J.r, & Nakhasi H.L. (2020). Essential role of neutrophils in the protective immune response induced by a live attenuated Leishmania vaccine. Journal of immunology (Baltimore, Md. : 1950), 205(12), 3333-3347.

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Splenocyte Immunophenotyping by Flow Cytometry

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Freshly prepared splenocytes were incubated in mouse Fc block (Rat anti-mouse CD16/32, BD Biosciences, catalog # 553142, 1:100) for 5 mins followed by incubation in antibodies for pan-leukocytes (Alexa Fluor 647 anti-mouse CD45, BioLegend, catalog # 103124, 1:100), B cells (PE-Cy7 Rat anti-mouse CD45R/B220, BD Biosciences, catalog # 552772, 1:100), and T cells (BV605 anti-mouse CD3, BioLegend, catalog # 100237, 1:100) in dark at 4°C for 30–45 mins, washed with PBS containing 2% FBS. Cells were stored on ice until analyzed using cytometer (CYTEK Northern Lights 3000, Fremont, CA).

Gaire J., Supper V., Montgomery D, & Simmons C.S. (2023). Spiny mice (Acomys) cells fail to engraft in NOD scid gamma. PLOS ONE, 18(5), e0286000.

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Identification of Th1, Th17, and Treg Cells

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HLN cells and splenocytes were stimulated with leukocyte activation cocktail (BD Pharmingen, San Jose, CA, USA). The cells were incubated with purified rat anti-mouse CD16/32 (BD Pharmingen) to block the Fc receptors (FcRs) and then stained with primary antibodies (BD Pharmingen) at 4°C for 30 min. Fluorescence-activated cell sorting (FACS) was performed using a BD Accuri™ C6 Plus (BD Bioscience, San Jose, CA, USA). Th1 cells were identified as CD4 and IFN-γ double-positive cells. Th17 cells were identified as CD4 and IL-17A double-positive cells. CD4 and forkhead box P3 (Foxp3) were used to identify Treg cells. FACS data were analyzed using FlowJo V10 software.

Zhang Y., Li C., Li S., Lu Y., Du S., Zeng X., Chen X, & Chen J. (2019). Dihydrotanshinone I Alleviates Crystalline Silica-Induced Pulmonary Inflammation by Regulation of the Th Immune Response and Inhibition of STAT1/STAT3. Mediators of Inflammation, 2019, 3427053.

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