Anti cd49b

Manufactured by BD

Anti-CD49b is a monoclonal antibody that binds to the CD49b antigen. CD49b is a cell surface receptor that plays a role in cell adhesion and signaling. The anti-CD49b antibody can be used for the identification and analysis of cells expressing the CD49b antigen.

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3 protocols using anti cd49b

Multiparametric Flow Cytometry Analysis of Immune Cell Subsets

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Surface and intracellular staining were performed as previously described [28 (link)]. Single immune cell populations from the spleen, lymph nodes or tumors were separated with a BD FACSAria II Cell Sorter. Flow cytometric analyses were performed with Flowjo (Tree Star). The following antibodies were used for cell staining: anti-CD3 (clone 145-2C11), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD49b (clone DX5), anti-CD11b(clone M1/70), anti-CD11c (clone HL3), anti-CD19 (clone 1D3), anti-CD25 (clone PC61), anti-CD69 (clone H1.2F3), anti-CD62L (MEL-14), anti-CD44 (clone IM7), anti-Foxp3 (clone FJK-16s), anti-Granzyme B (clone GB11), anti-CCR4 (clone 2G12), anti-CCR5 (clone HM-CCR5), anti-CXCR3 (clone CXCR3-173), NK1.1(clone PK136), anti-F4/80 (clone BM8), anti-Gr-1 (clone RB6-8C5), anti-interferon-γ (IFN-γ, clone XMG1.2), and anti-NK1.1 (clone PK136), CD4 blocking mAb (clone GK1.5), and CD8 blocking mAb (clone 53-6.7).
For detection of phosphorylated S6 proteins, cells from LNs cultured with PMA (10ng/ml) and Ionomycin (500ng/ml) at designated times were immediately fixed with phosflow Lyse/Fix buffer (BD Biosciences) and permeabilized by Phosflow Perm buffer (BD Biosciences). Cells were stained with the Alex488 conjugated antibody for S6P (Ser235,236) (D57.2. 2E; Cell Signaling Technology)

Rao E., Zhang Y., Zhu G., Hao J., Persson X.M., Egilmez N.K., Suttles J, & Li B. (2015). Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death. Oncotarget, 6(10), 7944-7958.

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Tumor Dissociation and Immune Cell Profiling

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Tumor tissues were washed in PBS, minced into small fragments, and incubated in collagenase solution (1 mg/ml collagenase IV in RPMI-1640) and DNase I (Sigma-Aldrich) at 37°C, 120 rpm for 2.5 hours. Dissociated cells were passed through a 40 μm strainer to achieve single cell suspensions followed by erythrocyte lysis. The following monoclonal antibodies were used in flow cytometry: Fixable Viability Stain 510, anti-CD45 (BV605; 30-F11), anti-CD3e (APC-Cy7; 145–2C11), anti-CD4 (FITC; RM4–5), anti-CD25 (PE-Cy7; PC61), anti-CD8a (PerCP-Cy5.5; 53–6.7),, anti-CD11c (PE-Cy7; HL3), anti-I-A/I-E (BV421; M5/114.15.2), anti-F4/80 (PE; T45–2342), anti-CD11b (FITC; M1/70), anti-CD49b (APC; DX), anti-Gr-1 (BV510), anti-Ly-6G (PE; 1A8), anti-Ly-6C (APC; AL-21) (all from BD Biosciences). Single cell suspensions were stained with 1 μg/sample fluorochrome-labeled antibodies for specific surface marker at 4 °C for 30 min in 100 μl PBS. Then the cells were fixed, permeabilized and stained for intracellular cytokines including anti-Foxp3 (BV421; MF23) and anti-IFN-γ (PE; XMG1.2) by using a fixation/permeabilization kit (BD Biosciences). Stained single-cell suspensions of tumor tissue were processed to flow cytometry using CytoFlex. The data was analysis by FlowJo v10 software. Gating strategies are displayed in supplementary figure 6.

Yang B., Li X., Fu Y., Guo E., Ye Y., Li F., Liu S., Xiao R., Liu C., Lu F., Huang J., Qin T., Han L., Peng G., Mills G.B., Sun C, & Chen G. (2021). MEK inhibition remodels the immune landscape of mutant KRAS tumors to overcome resistance to PARP and immune checkpoint inhibitors. Cancer research, 81(10), 2714-2729.

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Erythroid Cell Analysis by Flow Cytometry

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Erythroid cells were analyzed from the spleen and the bone marrow by flow cytometry. Cell suspensions were stained in phosphate-buffered saline (PBS) supplemented with sterile 5% fetal bovine serum (FBS). The following monoclonal antibodies were used for murine flow cytometric analysis: anti-Ter119-PE (BD Biosciences, 553673), anti-CD44-PE-Cy7 (BD Biosciences, 560599). The lineage (Lin) APC-conjugated antibodies were used: anti-Ly-6G (BD Biosciences, 560599), anti-Cd11c (BD Biosciences, 561119), anti-CD3 (BD Biosciences, 565643), anti-cd11b (BD Biosciences, 553312), anti-CD49b (BD Biosciences, 560628), anti-CD19 (BD Biosciences, 550992). Specifically, erythrocytes were defined as Lin⁻, Ter119⁺. This assay allows the separation of erythroid cells into distinct populations corresponding to (I) proerythroblasts, (II) basophilic, (III) polychromatic, (IV) orthochromatic cells and reticulocytes, and (V) RBCs (supplemental Figure 1). A minimum of 250 000 events were recorded for erythrocytes in spleen and bone marrow. For all the analyses, cells were acquired using the MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec) and the results analyzed with FlowJo software (Tree Star Inc, Ashland, OR).

Wake M., Palin A., Belot A., Berger M., Lorgouilloux M., Bichon M., Papworth J., Bayliss L., Grimshaw B., Rynkiewicz N., Paterson J., Poindron A., Spearing E., Carter E., Hudson R., Campbell M., Petzer V., Besson-Fournier C., Latour C., Largounez A., Gourbeyre O., Fay A., Coppin H., Roth M.P., Theurl I., Germaschewski V, & Meynard D. (2024). A human anti-matriptase-2 antibody limits iron overload, α-globin aggregates, and splenomegaly in β-thalassemic mice. Blood Advances, 8(8), 1898-1907.

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