Cd24 m1 69

Manufactured by BioLegend

CD24 (M1/69) is a cell surface glycoprotein expressed on a variety of cell types, including some hematopoietic cells and neural cells. It functions as an adhesion molecule and is involved in cell-cell interactions. This product is intended for research use only and its core function is to detect and characterize CD24-expressing cells.

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8 protocols using cd24 m1 69

Multiparametric Flow Cytometry of Immune Cells

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BM, splenocytes, and LN cells were stained with fluorescent conjugated Abs specific for CD4 (GK1.5, Tonbo Biosciences), CXCR5 (L138D7, BioLegend), PD1 (29F.1A12, BioLegend), CD38 (90, BioLegend), GL7 (GL7, BioLegend), B220 (RA3-6B2, BioLegend), CD93 (AA4.1, BioLegend), CD19 (1D3, Tonbo Biosciences), CD21 (7E9, BioLegend), CD23 (BEB4, BioLegend), CD24 (M1/69, BioLegend), CD62L (MEL-14, Tonbo Biosciences), CD25 (7D4, Tonbo Biosciences), CD43 (S7, BD Pharmigen), IgD (11-26C.2A, BioLegend), and IgM (DS-1, BD Pharmigen); Igκ (1050-02; Southern Biotech); T-bet (EBIO4B10, eBiosciences) and Bcl6 (K112-91, eBioscience); and BrdU (BU20A, BioLegend). Other reagents include Caspase3/7 (C10427, Invitrogen), ghost dye (13-0865-T100, Tonbo Biosciences), and NP-PE (N-5070-1; Biosearch Technologies). Cells were analyzed by flow cytometry as previously described (86 (link)). VLP Ab was a gift from Shaun Jackson. Intracellular Bcl6 staining to identify Tfh cells was performed as previously described (87 (link)). Intracellular staining for T-bet was performed as previously described (23 (link)).

Avalos A., Tietsort J.T., Suwankitwat N., Woods J.D., Jackson S.W., Christodoulou A., Morrill C., Liggitt H.D., Zhu C., Li Q.Z., Bui K.K., Park H, & Iritani B.M. (2022). Hem-1 regulates protective humoral immunity and limits autoantibody production in a B cell–specific manner. JCI Insight, 7(9), e153597.

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Multiparametric Flow Cytometry Analysis

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Cell suspensions from mouse liver, BM (one femur and tibia), spleen, thymus, mesenteric lymph nodes, and peritoneal cavity were processed as previously described (47 (link)), and data were acquired on a FACSCanto10c (BD) and analyzed with FlowJo Software (Tree Star).
The following anti-mouse antibodies were used: B220 (RA3-6B2, BioLegend), BAFFR (7H22-E16,BD), BP-1 (6C3, BioLegend), CD3e (145-2C11, eBioscience), CD4 (GK1.5, eBioscience), CD5 (53-7.3, BioLegend), CD8 (53-6.7, BioLegend), CD9 (MZ3, BioLegend), CD11b (M1/70, eBioscience), CD19 (6D5, 1D3, BioLegend), CD21/CD35 (7E9, BioLegend), CD23 (B3B4, BioLegend), CD24 (M1/69, BioLegend), CD25 (PC61, Biolgend), CD43 (S7, BioLegend), CD44 (IM7, BioLegend), CD45.1 (A20, BioLegend), CD45.2 (104, BioLegend), CD69 (H1.2F3, BioLegend), CD80 (16-10A1, BioLegend), CD86 (GL1, BioLegend), CD93 (AA4.1, BioLegend), DAPI (BIOTIUM), Gr-1 (RB6-8C5, eBioscience), IgDa (AMS-9.1, BioLegend), IgD (11-26c, BioLegend), IgMa (DS-1, MA-69, BioLegend), IgM (Il/41, RMM-1, BioLegend), TCRγδ (GL3, BioLegend), Live dead dye (Zombie Aqua Dye, BioLegend), Live dead dye (Zombie NIR, BioLegend), pERK (4B11B69, BD), pPLCγ2 (K86-1161, BD), Lin28b (AP1485C, ABGENT), and anti-rabbit IgG (BioLegend).

Xu X., Deobagkar-Lele M., Bull K.R., Crockford T.L., Mead A.J., Cribbs A.P., Sims D., Anzilotti C, & Cornall R.J. (2020). An ontogenetic switch drives the positive and negative selection of B cells. Proceedings of the National Academy of Sciences of the United States of America, 117(7), 3718-3727.

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Fluorochrome Antibody Staining Protocol

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Fluorochrome conjugates of antibodies against human Vβ11 (C21; Beckman Coulter), IFN-γ (4S.B3; Biolegend), CD4 (SK3; BD Biosciences), CD3 (HIT3a), CD8 (RPA-T8), IL-17 (64DEC17), and IL-4 (8D4-8), all from eBioscience, were used. Fluorochrome conjugates of antibodies against mouse CD24 (M1/69; Biolegend), B220 (RA3-6B2), CCR6 (140706), and IL-17 (TC11-18H10), all from BD Biosciences, CD4 (RM4-5), CD103 (2E7), Ki-67 (SolA15), IFN-γ (XMG1.2) and RORγt (AFKJS-9), all from eBioscience, were used.

Li S., Joseph C., Becourt C., Klibi J., Luce S., Dubois-Laforgue D., Larger E., Boitard C, & Benlagha K. (2014). Potential Role of IL-17-Producing iNKT Cells in Type 1 Diabetes. PLoS ONE, 9(4), e96151.

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Multicolor Flow Cytometry for Immune Cell Analysis

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The following anti-mouse antibodies, including Lineage cocktail (17A2; RB6-8C5, RA3-6B2; Ter-119; M1/70); CD117 (cKit, 2B8), Ly-6A/E (Sca-1, D7), CD3 (145-2C11), CD19 (6D5), B220 (RA3-6B2), Ly-6C (HK1.4), Ly-6G (1A8), CD45.1 (A20), CD24 (M1/69), and CX3CR1 (SA011F11) were purchased from Biolegend. Ki-67-FITC staining set was purchased from BD Biosciences. Mouse CCR2 APC-conjugated antibody was purchased from R&D Systems. Anti-myeloperoxidase (MPO) antibody (2D4) FITC was purchased from Abcam. Intracellular fixation and permeabilization solution kit was purchased from BD. Violet cell proliferation kit were purchased from Thermo Fisher Scientific. Cyclophosphamide monohydrate (CTX) was purchased from Tokyo Chemical Industry. 5-azacytidine was purchased from Selleckchem. Anti-mouse TNFa (XT3.11) mAb was purchased from Bio X Cell. Mouse IL6 neutralization mAb was purchased from Biolegend.

Ding Z.C., Aboelella N.S., Bryan L., Shi H, & Zhou G. (2021). The Monocytes That Repopulate in Mice After Cyclophosphamide Treatment Acquire a Neutrophil Precursor Gene Signature and Immunosuppressive Activity. Frontiers in Immunology, 11, 594540.

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Immunophenotyping and Protein Analysis

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The following antibodies were purchased from BioLegend, eBioscience, or BD Biosciences and used for flow cytometry or enrichment: FITC-Sirpα, PE/Cy7-Sirpα, PerCP/Cy5.5-B220, APC-EpCAM, PE-CD11c, PE/Cy7-CD11c, Brilliant Violet 605-CD11c, Pacific Blue-IA/IE, FITC-CD8α, Alexa700-CD8α, PE-V_α2, APC-V_α2, APC-V_β5, PE-V_β6, PE-Foxp3, Pacific Blue-Foxp3, PerCP/Cy5.5-Thy1.1, APC-TCRβ, PE-CD69, PE/Cy7-CD69, Brilliant Violet 605-CD4, APC-CD25, Brilliant Violet 605-CD25, Brilliant Violet 785-CD25, FITC-CD45RB, APC-ICAM1, PR-CD9, PE-CD81, APC-CD48, PE/Cy7-CD24, APC-CD24, PE-CD71, PE/Cy7-CD45, APC-streptavidin, Biotin-Thy1.2, Biotin-CD8β, Biotin-CD25, and Biotin-B220. Alexa647-CTxB subunit was purchased from Molecular Probes. For Western blotting, the following clones were used: MHCII β chain (Bett; homemade), CD48 (OX-78; Serotec), CD24 (M1/69; BioLegend), actin (A2066; Sigma), CD9 (LMC8; eBioscience), and CD81 (Eat2; BioLegend), and HRP-conjugated secondary antibodies (anti-rat, anti-rabbit, and anti-hamster) were purchased from Jackson ImmunoResearch or BIOSOURCE. Myriocin, MβCD, cholesterol, sphingomyelin, and 4-hydroxy-tamoxifen were purchased from Sigma.

Oh J., Perry J.S., Pua H., Irgens-Möller N., Ishido S., Hsieh C.S, & Shin J.S. (2018). MARCH1 protects the lipid raft and tetraspanin web from MHCII proteotoxicity in dendritic cells. The Journal of Cell Biology, 217(4), 1395-1410.

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Comprehensive Cellular Flow Cytometry Protocol

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For cellular flow cytometry, mAbs included the following: Thy1.2/CD90 (30-H12, BioLegend), TCRβ (H57, eBioscience), Vα2 (B20.1, BD), CD3ε (145-2C11, BioLegend), CD8α (53.6.7, BioLegend), CD8β (53-5.8, BioLegend), CD4 (GK1.5, BD Biosciences), CD69 (H1.2F3, BioLegend), CD24 (M1/69, BioLegend), and CD5 (53-7.3, BioLegend). Other binding reagents included the following: peanut agglutinin (PNA)-FITC (Sigma-Aldrich), streptavidin-PE and streptavidin-APC-Cy7 (BioLegend), H-2Kb-SIINFEKL (OVA) tetramer, and thymic leukemia antigen (TLA) tetramers (NIH tetramer core facility). For multiplex IP and PiSCES analysis, antimouse Abs are listed in table S1, whereas a similar table for antihuman Abs used in conjunction with Jurkat and JRT3 cell lines was previously published (40 (link)).

Neier S.C., Ferrer A., Wilton K.M., Smith S.E., Kelcher A.M., Pavelko K.D., Canfield J.M., Davis T.R., Stiles R.J., Chen Z., McCluskey J., Burrows S.R., Rossjohn J., Hebrink D.M., Carmona E.M., Limper A.H., Kappes D.J., Wettstein P.J., Johnson A.J., Pease L.R., Daniels M.A., Neuhauser C., Gil D, & Schrum A.G. (2019). The early proximal αβ TCR signalosome specifies thymic selection outcome through a quantitative protein interaction network. Science immunology, 4(32), eaal2201.

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Isolation and Purification of Thymocyte and Peripheral T Cell Subsets

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Subsets of thymocytes were isolated by cell sorting as previously described [54 (link)], after cell surface staining using CD4 (GK1.5), CD8 (53–6.7), CD3ε (145-2C11), and CD24 (M1/69) (all from Biolegend). DP cells were CD4+ CD8 int/hi; CD4 SP cells were CD4CD3 hi, CD24 int/lo. Peripheral subsets were isolated after pooling spleen and lymph nodes. T cells were enriched by negative isolation using Dynabeads (Dynabeads untouched mouse T cells, 11413D, Invitrogen). After surface staining for CD4 (GK1.5), CD8 (53–6.7), CD62L (MEL-14), CD25 (PC61) and CD44 (IM7), naïve CD4 + CD62LhiCD25-CD44lo were obtained by sorting (BD FACS Aria). Three cell isolations from independent mice were prepared for each of the three thymocyte subsets.

Äijö T., Huang Y., Mannerström H., Chavez L., Tsagaratou A., Rao A, & Lähdesmäki H. (2016). A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways. Genome Biology, 17, 49.

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T Cell Purification from Murine Tissues

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CD8 or CD4 T cells were purified from single-cell suspensions of spleen and lymph nodes from male and female mice aged 6–12 weeks by negative selection with biotinylated antibodies to CD4 (GK1.5) or CD8 (53-6.7) respectively, CD19 (6D5), B220 (RA3-6B2), CD11b (M1/70), CD49b (DX5), Ly-76 (Ter119), CD24 (M1/69) (all BioLegend) and CD11c (N418, Tonbo) and magnetic anti-biotin beads (MACSi Beads, Miltenyi Biotec) as previously described(15 (link)). Tregs were additionally depleted from CD4 T cells by adding 1:100 anti CD25 (PC61.5).

Au-Yeung B., Smith G.A., Mueller J.L., Heyn C.S., Jaszczak R.G., Weiss A, & Zikherman J. (2017). IL-2 modulates the TCR signaling threshold for CD8 but not CD4 T cell proliferation on a single cell level. Journal of immunology (Baltimore, Md. : 1950), 198(6), 2445-2456.

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