F4 80 buv395

Manufactured by BD

F4/80-BUV395 is a fluorescently-labeled antibody that binds to the F4/80 antigen, a cell surface glycoprotein expressed on mature macrophages and microglia in mice. This product is intended for use in flow cytometry applications to identify and characterize macrophage populations.

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F4/80 (149 citations)

3 protocols using f4 80 buv395

Liver Macrophage Phenotyping and Pyroptosis

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Animals were sacrificed and livers were collected as described above. Fractionated immune cells were collected. Cells were incubated with CD16/CD32 FcR-blocking antibody (BD Bioscience, 553142) on ice for 20 minutes. Live/Dead staining was performed using Zombie Aqua (Biolegend, 423101) with a 30-minute incubation on ice. HMΦ surface staining was performed using the following panel: APC-Cy7_CD45 (BioLegend, 103116), FITC_I-A/I-E (MHCII) (BioLegend, 107605), PerCP-Cy5.5_CD11b (BioLegend, 101228), BV510_Ly6G (BioLegend, 127633), BUV395_F4/80 (BD Biosciences, 565614), BV421_CCR2 (BioLegend, 150605), APC_Ly6C (BioLegend, 128016), PE-Cy7_CD206 (ThermoFisher, 25-2061-82), BV785_CD86 (BioLegend, 105043). HMΦ pyroptosis was performed using the following panel: FAM-LEHD-FMK (caspase-11 activity assay), APC-Cy7_CD45 (BioLegend, 103116), BV510_Ly6G (BioLegend, 127633), PerCP-Cy5.5_CD11b (BioLegend, 101228), Ly-6C_AF700 (BioLegend, 128024), BUV395_F4/80 (BD Biosciences, 565614), GSDMDC1_AF674 (Santa Cruz Biotechnology, sc-393581 AF647). Flow cytometric data was acquired using LSR-II Flow Cytometer (BD Bioscience). Mean fluorescent intensity (MFI) and population percentages were analyzed using FlowJo (Ashland, OR).

Drummer C I.V., Saaoud F., Jhala N.C., Cueto R., Sun Y., Xu K., Shao Y., Lu Y., Shen H., Yang L., Zhou Y., Yu J., Wu S., Snyder N.W., Hu W., Zhuo J.‘., Zhong Y., Jiang X., Wang H, & Yang X. (2023). Caspase-11 promotes high-fat diet-induced NAFLD by increasing glycolysis, OXPHOS, and pyroptosis in macrophages. Frontiers in Immunology, 14, 1113883.

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Comprehensive Immune Profiling of Tumor Cells

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In order to obtain optimal results, a minimum of 40 million tumor cells were blocked with anti-CD16/32 antibody then magnetically labeled using CD4 T cell isolation kit (Miltenyi Biotec Inc., 130-104-454). A minimum of 5 million CD4 positive cells were stained with CD4-APC (eBioscience, 17-0041-81) and then fixed and permeabilized using the Foxp3 staining kit (Thermofisher, 00-5523-00) and stained intracellularly with Foxp3-FITC (eBioscience, 11-5773-80). CD4 negative cells were stimulated using PMA-Ionomycin and Golgi stop then stained for surface markers CD8-BV650 (BD bioscience, 563822), Gr-1-APC-Cy7 (BD bioscience, 557661), CD11b-APC (Biolegend, 101211), CD206-BV421 (Biolegend, 141717), F4/80-BUV395 (BD bioscience, 565614) and fixed then permeabilized using the Foxp3 staining kit and stained intracellularly for IFN-γ-PE.

Ayoub M., Shinde-Jadhav S., Mansure J.J., Alvarez F., Connell T., Seuntjens J., Piccirillo C.A, & Kassouf W. (2019). The immune mediated role of extracellular HMGB1 in a heterotopic model of bladder cancer radioresistance. Scientific Reports, 9, 6348.

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Multiparametric Flow Cytometry for Immune Profiling

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First, single cell suspensions were incubated for 20 min in Fixable Viability Dye (FVD) eFluor™ 780 (eBioscience) in the dark on ice to exclude dead cells. After adding 1 µg TruStain™ FcX (Biolegend), immunostaining was performed on ice for 30 min (protected from light) with the following antibodies: CD45-AF700 (# 103,128), CD45-BV785 (# 103,149), Ly6C-APC (# 128,015), Ly6C-FITC (# 128,005), Ly6G-PE/Dazzle594 (# 127,648), CD11c-BV605 (# 117,334), CD8-PerCPCy5.5 (# 100,734), PD1-APC (# 135,210), CD62L-BV605 (# 104,438), CD44-FITC (# 103,005), CD19-FITC (# 115,506), CD19-PECy7 (# 115,520), PD1-BV421 (# 135,221) (all antibodies from Biolegend), and CD11b-BUV737 (# 612,801), F4/80-PE (# 565,410), Siglec-F-PErCP5.5 (# 565,526), F4/80-BUV395 (# 565,614), CD3-BUV395 (# 563,565), CD4-BUV496 (# 564,667), CD44-BUV737 (# 612,799), Siglec-F-PE (# 562,068) (all antibodies from BD Biosciences).
After staining, cells were washed and fixed using IC Fixation Buffer (eBioscience). Samples were either analyzed on a BD LSRFortessa™ or BD Canto II™ flow cytometer with FACSDiva software (BD Biosciences). Analyses and compensation were performed with Flowjo software (TreeStar). Fluorescence minus-one-samples were used to define gates. See Kienzl et al. [10] (link) (Supplementary material) for gating strategies.

Gruden E., Kienzl M., Hasenoehrl C., Sarsembayeva A., Ristic D., Schmid S.T., Maitz K., Taschler U., Hahnefeld L., Gurke R., Thomas D., Kargl J, & Schicho R. (2023). Tumor microenvironment-derived monoacylglycerol lipase provokes tumor-specific immune responses and lipid profiles. Prostaglandins, leukotrienes, and essential fatty acids, 196.

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