Cd16 32

Manufactured by BioLegend

Sourced in United States

CD16/32 is a lab equipment product that functions as a regulator of Fc gamma receptor (FcγR) expression. It is used in experimental research settings to study the role of FcγR in various cellular processes.

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Lab products found in correlation

Cd11b, by BioLegend (8 mentions) F4 80, by BioLegend (8 mentions) Granzyme b, by BioLegend (5 mentions) Cd11c, by BioLegend (5 mentions) Ifn γ, by BioLegend (5 mentions) Cd206, by BioLegend (5 mentions) Tnf α, by BioLegend (4 mentions) Foxp3, by BioLegend (4 mentions) Sca 1, by BioLegend (4 mentions) Software, by FlowJo (4 mentions) Cd45 apc cy7, by BioLegend (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Fixable viability dye efluor 506, by Thermo Fisher Scientific (3 mentions) Dnase 1, by Merck Group (3 mentions) Gallios flow cytometer, by Beckman Coulter (3 mentions) Interleukin 2 (il 2), by BioLegend (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Propidium iodide, by Thermo Fisher Scientific (3 mentions) Ly 6g ly 6c, by BioLegend (2 mentions) I a 1 e, by BioLegend (2 mentions)

68 protocols using cd16 32

Dextramer-Based Detection of Antigen-Specific T Cells

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The following H2-D^d dextramers were purchased from Immudex (Copenhagen, Denmark, and Fairfax, VA): IGPGRAFYTT (PE), IGPGRAFYT (APC), IGPGRAFYVV (PE), and IGPGRAFYV (APC). For dextramer staining, spleen cells (1–5 × 10⁶) were isolated and directly stained for CD3, CD4, CD8, CD16/32 (Biolegend), Yellow Viability or AquaBlue (Life Technologies) plus the indicated dextramers. For intracellular staining, single cell suspensions (10⁶ cells/μL) from spleen were isolated and stimulated with indicated peptides or ionomycin and phorbol 12-myristate 13-acetate (PMA), as previously described (27 (link)–29 (link)). Subsequently, cells were removed and stained for CD3, CD4, CD8, CD16/32, IL2, IFNγ (Biolegend) and TNFα (BD Pharmingen). Intracellular cytokine production was quantified using the previously described method (27 (link)–29 (link)). Stained cells were analyzed using a BD LSR II flow cytometer with FlowJo software.

Frey B.F., Jiang J., Sui Y., Boyd L.F., Yu B., Tatsuno G., Billeskov R., Solaymani-Mohammadi S., Berman P.W., Margulies D.H, & Berzofsky J.A. (2018). Effects of cross-presentation, antigen processing, and peptide binding in HIV evasion of T cell immunity. Journal of immunology (Baltimore, Md. : 1950), 200(5), 1853-1864.

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Multicolor Flow Cytometry of SVF Cells

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For surface markers detection, SVF cells were suspended in PBS containing 1% fetal bovine serum and incubated with Zombie dye (1:100, Cat#423106, Biolegend, USA) at room temperature for 7 min. Then cell suspension was incubated with CD16/32 (1:100, Cat#101302, Biolegend, USA) at room temperature for 7 min, followed by incubation with fluorochrome-conjugated antibodies (Supplementary Table 1) for 7 min at room temperature. For intracellular staining, samples were added with Monensin (1:1000, Cat#420701, Biolegend, USA) in the whole process to block the secretion of CCL5. The SVF cells were incubated with Zombie dye, blocked by CD16/32, and stained with fluorochrome-conjugated antibodies (Supplementary Table 1) against cell surface antigens. Then cells were fixed and permeabilized with cytofix/cytoperm buffer (Cat#554714, BD Bioscience, USA) for 45 min at 4 °C and then stained with fluorochrome-conjugated antibody against CCL5 for 30 min at 4 °C. Cells were washed by PBS twice and centrifuged at 500g for 5 min, and the cell pellet was suspended in 200 μL FACS Buffer. Samples were processed on a CYTEK flow cytometer and analyzed using FlowJo software.

Zhou H., Liao X., Zeng Q., Zhang H., Song J., Hu W., Sun X., Ding Y., Wang D., Xiao Y, & Deng T. (2023). Metabolic effects of CCL5 deficiency in lean and obese mice. Frontiers in Immunology, 13, 1059687.

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Multicolor Flow Cytometry Immunophenotyping

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IEC cell lines or digested cell suspensions were washed with FACS buffer (PBS containing 5% FBS and 2 mmol/L EDTA) and centrifuged. Cell pellets were incubated with fragment crystallizable (Fc) receptor blocker (CD16/32; BioLegend, San Diego, CA) at 4ºC for 15 minutes and stained with antibodies or the matching isotype control at 4ºC for 25 minutes in the dark. For staining of intracellular markers, the cells were incubated with antibodies against surface markers, fixed, and permeated with Perm/Wash Buffer (BD Biosciences, Franklin Lakes, NJ) before intracellular staining. The following antibodies were used: CD45 (Brilliant Violet 711, 30-F11; BioLegend), CD11b (allophycocyanin, M1/70; BioLegend), F4/80 (phycoerythrin, BM8; BioLegend), CD11c (PE, N418; BioLegend), CD206 (Alexa Fluor 488, C068C2; BioLegend), MHCII (FITC, M5/114.15.2; BioLegend) CD4 (FITC, GK1.5; BioLegend), CD8 (PE/Cy5, SK1; BioLegend), and Gr-1 (PE/Cy7, RB6-8C5; BioLegend). Analysis or cell sorting was performed with the Aria Fusion Cell sorter and Cell Analyzer (BD Biosciences). The acquired data were analyzed by FlowJo software.

Xie M., Mak J.W., Yu H., Cheng C.T., Chan H.C., Chan T.T., Lau L.H., Wong M.T., Ko W.H., Jiang L, & Yao X. (2022). TM9SF4 Is a Crucial Regulator of Inflammation and ER Stress in Inflammatory Bowel Disease. Cellular and Molecular Gastroenterology and Hepatology, 14(2), 245-270.

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Comprehensive Immune Cell Phenotyping

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Data were analyzed with FlowJo software. Antibodies to mouse CD45 (catalog no. 103133 and 103108), CD3 (catalog no. 100210), CD4 (catalog nos. 100434 and 100446), CD8α (catalog no. 100714), CD86 (catalog nos. 105008 and 105026), CD11c (catalog no. 117346), CD11b (catalog no. 101210), CD206 (catalog no. 141721), CD25 (catalog no. 101904), CD62L (catalog no. 104406), CD44 (catalog no. 103018), CD80 (catalog no. 104718), CD40 (catalog no. 102906), F4/80 (catalog no. 123118), Ly-6G/Ly-6C (catalog no. 108417), FOXP3 (catalog no. 126408), NK-1.1 (catalog no. 108710), I-A/I-E (catalog no. 107608), IFN-γ (catalog no. 505830), TNF-α (catalog no. 506308), IL-4 (catalog no. 504124), granzyme B (catalog no. 515406), and CD16/32 (catalog no. 101320) were obtained from BioLegend. Phycoerythrin-conjugated tetramer was from National Institutes of Health (NIH) Tetramer Core Facility. All reagents were used according to the manufacturer’s specifications.

Cheng F., Su T., Zhou S., Liu X., Yang S., Lin S., Guo W, & Zhu G. (2023). Single-dose injectable nanovaccine-in-hydrogel for robust immunotherapy of large tumors with abscopal effect. Science Advances, 9(28), eade6257.

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Frozen Tissue Immunofluorescence Imaging

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Tissues were embedded in OCT (Thermo Scientific), frozen on dry ice for 10 min and stored at −80°C until processing. Frozen tissues were cut to 8μm using a Microm HM550 cryostat (Thermo Scientific). Slides were fixed with acetone (cat. A18–4, Fisher Chemical) at −20°C for 10 min and washed with PBS (Gibco) for 3 times. PAP pen (MilliporeSigma) was used to circle the sections and blocking buffer (3% FBS, Hyclone, Fisher) was added onto the slides and incubated in a moist chamber for over 1h. Slides were washed with PBS for 3 times and incubated with fluorochrome-conjugated antibodies against B220 (RA3–6B2), CD4 (RM4–5), CD73 (TY/11.8), GL7 (GL7), (all from Biolegend); kappa chain (187.1) (from BD Biosciences) and C3c (polyclonal, Nordic MUbio) at RT for 1h. CD16/32 (93, Biolegend) was used for Fc Block prior to Ig labeling for 30 minutes at RT. DAPI was used for nuclear counterstaining. Slides were imaged using a LSM 880 Confocal or Zeiss Axio Observer (Zeiss) microscope.

Zhu J., Hay A.N., Potter A.A., Richwine M.W., Sproule T., LeRoith T., Wilson J., Hasham M.G., Roopenian D.C, & Leeth C.M. (2020). Abrogated AID function prolongs survival and diminishes renal pathology in the BXSB mouse model of SLE. Journal of immunology (Baltimore, Md. : 1950), 204(5), 1091-1100.

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Multiparametric Immune Cell Profiling

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MLN and spleen were aseptically harvested followed by preparation of a single cell suspension. The cells were passed through a 40 µM BD strainer. 1 million cells were taken and blocked using CD16/32 (Biolegend-101302) antibody for 10 min on ice. The cells were surface stained with fluorescently labeled antibodies for CD45.2 (Biolegend-109824), Ly6G (Biolegend-127614), CD11c (Biolegend-117311), CX3CR1 (Biolegend-149006), CD11b (Biolegend-101251), CD206 (141705), CD301 (Biolegend-145710). Fluorescently labeled cells were washed twice with PBS and acquired on BD FACS Canto II and BD FACS Verse (BD Biosciences, USA). The acquired data were then analyzed using FlowJo^TM 10 software.

Rana S., Maurya S., Mohapatra G., Singh S., Babar R., Chandrasekhar H., Chamoli G., Rathore D., Kshetrapal P, & Srikanth C.V. (2021). Activation of epigenetic regulator KDM6B by Salmonella Typhimurium enables chronic infections. Gut Microbes, 13(1), 1986665.

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Visualizing Brain Vasculature in Mice

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Mice were subjected to deep anesthesia, and 200 μg of FITC‐conjugated isolectin B4 (Vector Laboratories Burlingame, CA, USA) were injected intracardially. The animals were then killed, and brain explants were established and incubated on cell culture inserts for at least 1 h. Alternatively, explants were incubated for 10 min with CD16/32 (1:300 dilution; BioLegend, San Diego, CA) to block IgG Fc receptors II/III, washed with PBS, and stained with phycoerythrin (PE)‐ or FITC‐conjugated antibodies to mouse CD31 (1:300, BioLegend) or to mouse Sca1 (1:300, BioLegend). Images were acquired with an FV10i confocal microscope (Olympus).

Minami N., Maeda Y., Shibao S., Arima Y., Ohka F., Kondo Y., Maruyama K., Kusuhara M., Sasayama T., Kohmura E., Saya H, & Sampetrean O. (2017). Organotypic brain explant culture as a drug evaluation system for malignant brain tumors. Cancer Medicine, 6(11), 2635-2645.

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Comprehensive Erythroblast Immunophenotyping

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Bone marrow cells were immunostained and analyzed on an LSRII flow cytometer (BD Biosciences). Fluorochrome-conjugated anti-mouse Ter119, CD45, CD44, CD71, CD117/c-Kit, Sca1, CD150, CD48, CD105, CD41 and CD16/32 antibodies were purchased from Biolegend. Cell death and apoptosis were analyzed using Annexin V (Biolegend, #640906) and DAPI (Thermo Fisher, D1306). For PI staining, bone marrow cells were stained with surface markers and then fixed with 70% ethanol for 15 min. Cells were washed and treated with RNase A (10ug/mL) at 37°C for 15 min. A final concentration of 50ug/mL PI were administered and cells were analyzed by flow.
To isolate erythroblasts, CD45 cells depletion was performed (CD45 MicroBeads, Miltenyi Biotec) and CD45-negative cells were then stained with CD45, TER119 and CD44 and sorted using a BD FACS Aria I or a BD FACS Aria III (BD bioscience)

Tu Z., Fan C., Davis A.K., Hu M., Wang C., Dandamudi A., Seu K.G., Kalfa T.A., Lu Q.R, & Zheng Y. (2022). Autism-associated chromatin remodeler CHD8 regulates erythroblast cytokinesis and fine-tunes the balance of Rho GTPase signaling. Cell reports, 40(2), 111072.

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Immune Cell Analysis in Musculoskeletal Tissue

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To analyze immune cells in the musculoskeletal tissues at 3 and 7 dpi, ipsilateral feet were perfused extensively, skinned, disjointed from the tibia, and digested in RPM11640 medium supplemented with 10% FBS, 15 mM HEPES, 0.5 mg/ml of collagenase (Sigma, C0130) and 10 ug/ml of DNase I (Sigma, D5025) for 1 h at 37°C. Digested tissue was passed through a 70 μm strainer, and cells were separated by centrifugation and washed with PBS supplemented with 2% FBS and 2 mM EDTA. Cells were stained with a Fixable Viability Dye eFluor 506 (eBioscience, 65-0866-14) and incubated with the following antibodies for 1 h at 4°C: CD16/32 (Biolegend, 101301, 1:200) CD11b PE-Dazzle 594 (BioLegend, 101256, 1:200), Ly6G PerCP-Cy5.5 (Biolegend, 127616, 1:400), Ly6C Pacific Blue (BioLegend, 128014, 1:200), F4/80 APC (Thermo Fisher, 17-4801-82, 1:200), CD11c PE-Cy7 (BD Biosciences, 558079, 1:200), I-A/I-E (MHC class II) Alexa Fluor 700 (BioLegend, 107622, 1:200), NK-1.1 PE (BioLegend, 108708, 1:200), CD3 BUV737 (BD Biosciences, 564380, 1:200), CD8 PerCP-Cy5.5 (BioLegend, 100734, 1:200), CD4 BV786 (BioLegend, 100552, 1:200), B220 BV711 (BioLegend, 103255, 1:200). Cells were analyzed using a BD X20 Fortessa flow cytometer, and all data were processed using FlowJo software (FlowJo, LLC).

Zhang R., Earnest J.T., Kim A.S., Winkler E.S., Desai P., Adams L.J., Hu G., Bullock C., Gold B., Cherry S, & Diamond M.S. (2019). Expression of the Mxra8 Receptor Promotes Alphavirus Infection and Pathogenesis in Mice and Drosophila. Cell reports, 28(10), 2647-2658.e5.

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Fluorescent Labeling and Cell Staining

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Human serum albumin (HSA), 2-(N-Morpholino) ethanesulfonic acid (MES), fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. MTX (≥98%), sodium dodecyl sulfate (SDS, ≥98%), dimethylsulfoxide (DMSO, ≥99%) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. Sodium hyaluronate (5 kDa) was purchased from Bloomage Freda Biopharm Co., Ltd., (Jinan, China). Paraformaldehyde (4%) was obtained from Beyotime Biotechnology Co., Ltd. Imiquimod cream (5%) was from Mingxin Pharmaceuticals (Sichuan, China). CD16/32, APC-Cy7-CD45, FITC-CD3, Qdot-605-CD11c were bought from BioLegend Inc. RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc.

Wang H., Zhao Z., Wu C., Tong X., Shi Y, & Chen S. (2022). Microneedle Patch Delivery of Methotrexate-Loaded Albumin Nanoparticles to Immune Cells Achieves a Potent Antipsoriatic Effect. International Journal of Nanomedicine, 17, 3841-3851.

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