Anti cd36

Manufactured by BioLegend

Sourced in United States

Anti-CD36 is a laboratory reagent used for the detection and analysis of the CD36 protein, which is a cell surface receptor involved in various biological processes. This product can be used in flow cytometry, immunohistochemistry, and other research applications to identify and study cells expressing CD36.

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5 protocols using anti cd36

Lipid Profiling of Tumor-Associated Macrophages

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Single-cell suspensions from surgically resected CLM tissues from four patients were obtained as described in the previous paragraph. To assess expression of lipid transporters (ABCA1 and ABCG1), ApoE, and CD36, the following antibodies were included in the cocktail: anti-ABCA1 (Invitrogen; rabbit polyclonal), anti-ABCG1 (Novus Biologicals; rabbit polyclonal), anti-ApoE (BioLegend; clone E6D7), and anti-CD36 (BioLegend; clone 5–271). Detection of ABCG1 and ApoE was performed by intracellular staining with BD Cytofix/Cytoperm Solution (BD Biosciences). Cells were acquired on a FACSAria III (BD Biosciences) following the gating strategy shown in Fig. S2.
For lipid staining, cells were stained with the same cocktail used for cell sorting and then incubated with HCS LipidTOX Deep Red Neutral Lipid Stain (ThermoFisher; dilution 1:200) in PBS for 30 min at room temperature. Cells were immediately washed with PBS and acquired by flow cytometer. Lipid content of L-TAMs and S-TAMs was evaluated as mean fluorescence intensity (MFI) of the dye.
To test lipid uptake, cells were incubated with 0.8 µM of the fluorescent dye BODIPY 558/568 C12 (ThermoFisher) in PBS containing 0.1% BSA (fatty acid–free) for 1 min at 37°C. Cells were immediately washed with cold PBS containing 0.2% BSA and acquired by flow cytometer. Lipid uptake of L-TAMs and S-TAMs was evaluated as MFI of the dye.

Donadon M., Torzilli G., Cortese N., Soldani C., Di Tommaso L., Franceschini B., Carriero R., Barbagallo M., Rigamonti A., Anselmo A., Colombo F.S., Maggi G., Lleo A., Cibella J., Peano C., Kunderfranco P., Roncalli M., Mantovani A, & Marchesi F. (2020). Macrophage morphology correlates with single-cell diversity and prognosis in colorectal liver metastasis. The Journal of Experimental Medicine, 217(11), e20191847.

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Comprehensive Antibody Panel for Cell Analysis

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Primary antibodies used in this study include anti-DYKDDDDK Tag (CST, D6W5B, # 15009), anti-CD36 (BioLegend, # 336206), anti-KIR3DL1 (BioLegend, # 312716), anti-anti-KIR3DL2 (R&D, # FAB2878A), anti-anti-KIR3DL3 (R&D, # FAB8919r), anti-KIR2DL2/L3 (BioLegend, # 312612), anti-CD122 (BioLegend, # 105912), anti-CD5 (BioLegend, # 364016), anti-CD25 (BioLegend, 302610), anti-CD272 (BioLegend, # 344510), anti-CD2 (BioLegend, # 300214), anti-CD28 (BioLegend, # 302912), anti-CD80 (BioLegend, # 305219), anti-CD45 (BioLegend, # 304012), anti-IL6ST (BioLegend, # 362006), anti-CD276 (BioLegend, # 351006), anti-CD47 (BioLegend, # 323124). These antibodies were used at 1:100 dilution in MACS staining buffer (Miltenyi).

Siepe D.H., Henneberg L.T., Wilson S.C., Hess G.T., Bassik M.C., Zinn K, & Garcia K.C. (2022). Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform. eLife, 11, e81398.

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T cell Proliferation and Activation Assay

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Isolated T cells were labeled with carboxy fluoroscein succinimidyl ester (CFSE) fluorescent dye (Cat# C1031, Beyotime), co-cultured with Panc02 cells at a ratio of 6:1 (CD3⁺T: Panc02 cells), and added OVV, OVV-GPX4 viral supernatant (MOI = 10) or PBS. After 48 hours, the proliferation of T cells was tested by flow cytometry. In other parallel experiments, T cells were co-incubated with OVV, OVV-GPX4 viral supernatant (MOI = 10) or PBS for 48 h. T cells suspension was stained with the following antibodies: anti-CD8(Cat# 100706, Biolegend), anti-CD25(Cat# 102021, Biolegend), anti-CD36(Cat# 102611, Biolegend) and Bodipy 581/591 C11 (Cat# D3861, Invitrogen).

Wei W., Tian L., Zheng X., Zhong L., Chen Y., Dong H., Zhang G., Wang S, & Tong X. (2024). Expression of GPX4 by oncolytic vaccinia virus can significantly enhance CD8+T cell function and its impact against pancreatic ductal adenocarcinoma. Oncoimmunology, 13(1), 2322173.

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Murine Dendritic Cell Lipid Uptake

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Murine DCs were surface stained for 30 mins in the dark at 4°C with fluorescently labelled antibodies against CD11c (clone N418, BD), CD4 (clone RM4-5, BD), CD8α (clone 53–6.7, BD), GR1 (clone RB6.8C5, BD), B220 (clone RA3.6B2, BD), CD40 (Biolegend, California, USA), MHC class-I (Biolegend) and MHC class-II (Biolegend). For intracellular lipid quantification, cells were incubated for 15 mins at 4°C in the dark with BODIPY 493/503 (Invitrogen).
For staining molecules associated with lipid uptake, cells were fixed in 1% paraformaldehyde (Sigma-Aldrich) and permeabilised with 0.1% saponin (Sigma-Aldrich). Cells were incubated with fluorescently labelled anti-CD36 (Biolegend), anti-CD68 (Biolegend), LRP-1 (Santa Cruz, California, USA), LPL (Abcam, Cambridge, UK) and VLDLr (Abcam) in the dark for 30 mins at 4°C. Before staining, mouse IgG₁ antibodies (LPL and VLDLr) were directly conjugated to zenon reagents (Invitrogen) as per manufacturer’s instructions. Following LRP-1 staining, cells were sequentially incubated with anti-goat biotin (Dako, California, USA) and streptavidin-V500 (BD) in the dark for 30 mins at 4°C.
Following staining, samples were washed twice and resuspended in FACS buffer for acquisition on a FACSCanto II (BD) followed by analysis using FlowJo V10 software (Treestar, Oregon, USA). Isotype-matched antibodies were used as controls.

Gardner J.K., Mamotte C.D., McGonigle T., Dye D.E., Jackaman C, & Nelson D.J. (2014). Lipid-laden partially-activated plasmacytoid and CD4−CD8α+ dendritic cells accumulate in tissues in elderly mice. Immunity & Ageing : I & A, 11, 11.

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Multimarker Phenotyping of Microglia

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Microglia were stained with Ghost Dye violet 510 (1:1,000; Tonbo Biosciences) to exclude dead cells followed by Fc receptor blocking using an anti-CD16/CD32 antibody (1:100; BD Biosciences) to avoid nonspecific staining. Appropriate microglial surface markers were used for staining, including anti-CD11b (1:100; BioLegend), anti-CD45 (1:100; BioLegend), anti-CD11c (1:50; BioLegend), anti-CD36 (1:100; BioLegend), anti-CD209a (1:100; BioLegend), anti–αV-integrin (1:50; BioLegend), anti–β3-integrin (1:50; BioLegend), anti-CD86 (1:100; BioLegend), and anti–MHC II (1:100; BioLegend), followed by fixation and permeabilization for subsequent intracellular staining with anti-OPN (1:10; R&D Systems) and anti–TNF-α (1:50; BioLegend) and intranuclear staining with anti–Ki-67 (1:100; BioLegend).

Shen X., Qiu Y., Wight A.E., Kim H.J, & Cantor H. (2022). Definition of a mouse microglial subset that regulates neuronal development and proinflammatory responses in the brain. Proceedings of the National Academy of Sciences of the United States of America, 119(8), e2116241119.

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