F4 80 alexa fluor 488

Manufactured by BioLegend

Sourced in United States

F4/80-Alexa Fluor 488 is a fluorescence-labeled antibody that binds to the F4/80 antigen, which is expressed on the surface of macrophages and other mononuclear phagocytes. The Alexa Fluor 488 dye allows for detection and analysis of F4/80-positive cells using flow cytometry or other fluorescence-based methods.

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F4/80 antibody conjugated to Alexa Fluor 488 Anti-F4/80 Alexa Fluor-488 and anti-CD11b APC/Cy7 Alexa Fluor 488 anti-mouse F4/80 Alexa Fluor 488-conjugated F4/80 Anti‐F4/80‐Alexa‐Fluor‐488 (BM8), Alexa Fluor 488 anti-mouse F4/80 antibody Alexa Fluor 488 F4/80 Alexa 488

7 protocols using f4 80 alexa fluor 488

Macrophage Migration Assay with Activin A

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Six hundred microliter of macrophage serum‐free medium (M‐SFM, Invitrogen) containing recombinant human activin A (R&D Systems, Minneapolis, MN) at various concentrations was placed into the lower wells of transwell plates (Corning Incorporated, Corning, NY) with 6.5 mm diameter inserts and 8.0 μm pore size. Macrophages (10⁵ per well, in duplicates) were seeded in 100 μl of M‐SFM (with or without activin A) on the inserts. After 24 h, the inserts were removed from the plate and the upper side was cleaned with cotton swabs to remove non‐migrated cells. Following fixation in cold 100% methanol for 10 min, inserts were stained with only Hoechst or together with F4/80‐Alexa Fluor488 (Biolegend) for 1–3 h. After washing with PBS, membranes from inserts were cut out, placed on the glass slides, and mounted with Mowiol. The number of migrated cells was counted by blinded investigators in five 10× or 20× microscope fields and normalized to control (M‐SFM).

Antsiferova M., Piwko‐Czuchra A., Cangkrama M., Wietecha M., Sahin D., Birkner K., Amann V.C., Levesque M., Hohl D., Dummer R, & Werner S. (2016). Activin promotes skin carcinogenesis by attraction and reprogramming of macrophages. EMBO Molecular Medicine, 9(1), 27-45.

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Prostate Cancer Xenograft Immune Profiling

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Using a Johns Hopkins Animal Care and Use Committee approved protocol, adult athymic nude mice were inoculated subcutaneously in the flank with the LNCaP human prostate cancer cell lines in 200 μL of Matrigel. Mice were divided into two groups, and the treatment group was implanted with 2 one cm long silastic implants filled with testosterone as described previously (6 ). Tumors were harvested 2- and 4-days post-treatment and fixed in 10% buffered formalin and processed for IHC and H&E staining. NK cells were identified using antiCD57-PE (Santa Cruz Biotechnology) and antiCD49b-FITC (Santa Cruz Biotechnology) antibodies. F4/80-Alexafluor 488 and Ly-6G- Alexafluor 488 antibodies (Biolegend) were used to stain the macrophages and neutrophils cells respectively. Stained sections were imaged using Zeiss LSM 700 laser confocal microscope. Images were analyzed using the image processing package Fiji. NK cells, macrophages and neutrophils were counted per field from at least five fields and plotted as mean values.

Kumar R., Mendonca J., Owoyemi O., Boyapati K., Thomas N., Kanacharoen S., Coffey M., Topiwala D., Gomes C., Ozbek B., Jones T., Rosen M., Dong L., Wiens S., Brennen W.N., Isaacs J.T., De Marzo A.M., Markowski M.C., Antonarakis E.S., Qian D.Z., Pienta K.J., Pardoll D.M., Carducci M.A., Denmeade S.R, & Kachhap S.K. (2021). Supraphysiological testosterone induces ferroptosis and activates immune pathways through nucleophagy in prostate cancer. Cancer research, 81(23), 5948-5962.

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Characterization of Immune Cell Populations

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Therapeutic anti-PD-1 (BE0146) and isotype control antibody (BE0089) were produced by BioXcell. Antibodies used for flow cytometry were purchased from eBioscience (Anti-mouse CD3ε FITC, CD11b eFluor 660) and Biolegend (Anti-mouse CD8a Alexa Fluor® 647, NK 1.1 Alexa Fluor® 647, Ly-6G/Ly-6C (Gr-1) Alexa Fluor® 488, CD19 FITC, CD45R/B220 Alexa Fluor® 647, F4/80 Alexa Fluor® 488, CD206 Alexa Fluor® 647, FOXP3 Alexa Fluor® 488, CD4 Alexa Fluor® 647, CD11c Alexa Fluor® 488, I-A/I-E Alexa Fluor® 647, CD16/32).

Hu H.J., Liang X., Li H.L., Wang H.Y., Gu J.F., Sun L.Y., Xiao J., Hu J.Q., Ni A.M, & Liu X.Y. (2021). Enhanced anti-melanoma efficacy through a combination of the armed oncolytic adenovirus ZD55-IL-24 and immune checkpoint blockade in B16-bearing immunocompetent mouse model. Cancer Immunology, Immunotherapy, 70(12), 3541-3555.

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Polarization of Bone Marrow-Derived Macrophages

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To measure the effect of shAcsl1 on the polarization of BMDMs, cells were exposed to vehicle or 1 × 10⁹ TU/mL shAcsl1 lentiviruses for three days. In some experiments, cells were treated with 5.5 (normal) or 25 mM (high) endotoxin-free d-glucose (Sigma Aldrich) before analysis. Briefly, cells were incubated with fluorescence-labeled antibodies (CD11b-APC, F4/80-Alexa Fluor 488, and CD86-PE or CD206-PE, Biolegend, USA) for 30 min. The cells were then washed three times with FACS buffer, followed by analysis using the BD FACS Calibur™. Flow cytometry data were analyzed using FlowJo software.

Wang T., Dong Y., Yao L., Lu F., Wen C., Wan Z., Fan L., Li Z., Bu T., Wei M., Yang X, & Zhang Y. (2022). Adoptive transfer of metabolically reprogrammed macrophages for atherosclerosis treatment in diabetic ApoE−/- mice. Bioactive Materials, 16, 82-94.

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Multicolor Flow Cytometry Profiling of Pancreatic Immune Cells

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Single cell suspensions were prepared from resected pancreata using a gentleMACS Octo Dissociator with the Tumor Dissociation Kit as per the manufacturers’ instructions. Levels of the following cell surface markers were directly measured by flow cytometry on a BD FACSCanto (BD Biosciences) in two separate groups as noted and analyzed using FlowJo Software (Tree Star). Group 1: CD45 (APC; BioLegend), CD11b (PE/Cy7; BioLegend), Gr1 (PerCP; BioLegend), CD11c (PE; BioLegend), F4/80 (Alexa Fluor^® 488; BioLegend), Fixable Viability Dye (eFluor™ 780; eBioscience) Group 2: CD45 (APC; BioLegend), Th1.2 (Alexa Fluor^® 488; BioLegend), IgMa (PE; BD Pharmingen), CD4 (PE/Cy7; BioLegend), CD8a (PerCP; BD Pharmingen), Fixable Viability Dye (eFluor™ 780; eBioscience).

Nevler A., Muller A.J., Sutanto-Ward E., DuHadaway J.B., Nagatomo K., Londin E., O’Hayer K., Cozzitorto J.A., Lavu H., Yeo T.P., Curtis M., Villatoro T., Leiby B.E., Mandik-Nayak L., Winter J.M., Yeo C.J., Prendergast G.C, & Brody J.R. (2018). Host IDO2 gene status influences tumor progression and radiotherapy response in KRAS-driven sporadic pancreatic cancers. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(2), 724-734.

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Multicolor Flow Cytometry Panel

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Fc Block (20 m g/mL; BD Biosciences) was used to block cell-surface antigens. After blocking cells were stained with fluorophore conjugated antibodies or isotype control antibodies. Fluorophore-conjugated primary antibodies were purchased from BioLegend: F4/80-Alexa Fluor 488, CD11b-PerCP/Cy5.5, CD11c-phycoerythrin, and CD206-Alexa Fluor 647. After incubation with antibodies, cells were washed and centrifuged, re-suspended in a washing buffer, and analyzed on a FACSCalibur using FlowJo 10.0.6.

Lin Y.W., Montassier E., Knights D, & Wei L.N. (2016). Gut microbiota from metabolic disease-resistant, macrophage-specific RIP140 knockdown mice improves metabolic phenotype and gastrointestinal integrity. Scientific Reports, 6, 38599.

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Naja mossambica mossambica Cardiotoxin Extraction

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Cardiotoxin (CTX) from Naja mossambica mossambica was purchased from Sigma Aldrich (St. Louis, MO, USA). For immunohistochemical staining, Pax7, bromodeoxyuridine (BrdU) and myosin heavy chain (MF20) antibodies were purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA). Ly6b.2 (clone 7/4) and CD11b were purchased from AbD Serotec (Raleigh, NC). For fluorescence-activated cell sorting (FACS), propidium iodide (PI), CD31-APC, CD45-APC, Sca1-PerCP-Cy5.5, Biotinylated-Vcam1; PE/Cy7-streptavidin, Ly6G/C-PE-Cy7, F4/80-Alexa Fluor 488, and CD206-PE were purchased from BioLegend (San Diego, CA). Calcein Blue Viability Dye was purchased from eBioscience (San Diego, CA).

Shi H., Gatzke F., Molle J.M., Lee H.B., Helm E.T., Oldham J.J., Zhang L., Gerrard D.E, & Bennett A.M. (2015). Mice lacking MKP-1 and MKP-5 Reveal Hierarchical Regulation of Regenerative Myogenesis. Journal of stem cell and regenerative biology, 1(1), 1-7.

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