Cd49b apc

Manufactured by BioLegend

Sourced in United States

CD49b-APC is a fluorescently-labeled monoclonal antibody that binds to the CD49b antigen. CD49b, also known as the integrin alpha 2 subunit, is a cell surface receptor involved in cell-cell and cell-matrix interactions. The APC (Allophycocyanin) fluorescent label allows for the detection of cells expressing CD49b using flow cytometry.

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Close spelling variants of this product

APC anti‐mouse CD49b (4 citations) Anti-CD49b-APC (2 citations) CD49b-APC (clone DX5, (2 citations) APC-rat anti-mouse CD49b (2 citations)

6 protocols using cd49b apc

Tumor Immune Landscape Analysis

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After the mice were sacrificed, the tumors were collected and digested in dissociation buffer [mixture of deoxyribonuclease (50 μg mL^–1), hyaluronidase (100 μg mL^–1), and collagenase IV (2 mg mL^–1)] at 37 °C for 1 h with gentle shaking. The cell suspension was passed through a 70 μm cell strainer and dispersed in PBS buffer to form single-cell suspension. For T lymphocytes detection, the single-cell suspension was stained with antibodies against CD3, CD4 and CD8 to mark the helper (CD3⁺ and CD4⁺) and cytotoxic (CD3⁺ and CD8⁺) T cells. The tumor-cell suspension was stained with antibodies against CD11b, F4/80, CD86, and CD206 to analyze M1-like (CD11⁺ and CD86⁺) and M2-like (F4/80⁺ and CD206⁺) macrophages. The mice’s blood and spleen were also collected for lymphocyte extraction using lymphocyte isolation kit (Solarbio) according to the manufacturer’s manual. NK cells (CD3⁻, CD49b⁺), γδ T cells (CD3⁺, TCRβ⁻), and memory T cells (CD8⁺, CD44^high, CD62L^low) were analyzed using flow cytometer. The anti-mouse of CD3-PE (clone: 17A2), CD4-APC-cy7 (clone: GK1.5), CD8a-FITC (clone: 53-6.7), CD11b-PE (clone: M1/70), F4/80-APC (clone: BM8), CD86-PE-Cy7 (clone: GL-1), CD206-FITC (clone: C068C2), CD49b-APC (clone: HMα2), TCRβ-FITC (clone: H57-597), CD44-PE (clone: IM7), and CD62L-APC-Cy7 (clone: MEL-14) were purchased from Biolegend.

Wang Y., Han Y., Yang C., Bai T., Zhang C., Wang Z., Sun Y., Hu Y., Besenbacher F., Chen C, & Yu M. (2024). Long-term relapse-free survival enabled by integrating targeted antibacteria in antitumor treatment. Nature Communications, 15, 4194.

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Multiparametric Flow Cytometry Analysis

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Venus expression was detected directly. Surface receptor expression was analyzed using antibodies CD45-AF700 (1:400; BioLegend, cat. #103127), CD19-PE (1:200; eBiosciences, cat. #12–0193), GR1-APC-Cy7 (1:400; BD Biosciences, cat. # 557661), CD11B-PerCP-Cy5.5 (1:500; eBioscience, cat. #45-0112-80), TER119-BV421 (1:400; BD Horizon, cat. #563998), CKIT-APCeFluor780 (1:800; eBioscience, cat. #47-1171-80), FCɛR1α-PE (1:200, MAR-1; BioLegend, cat. #134308), CKIT-BV421 (1:200; BD Biosciences, cat. #562609), and CD49B-APC (1:100; BioLegend, cat. #108909) for murine cells and CD41-PE (1:50; BioLegend, cat. #303705), FCɛRIα-PE (1:50; BioLegend, cat. #334610), and CKIT-PECy7 (1:50; BioLegend, cat. #313212) for human cells. Dead cells were excluded with Hoechst 33342 (Life Technologies). Cells were sorted/analyzed on FACSAria IISORP (BD Biosciences) with FlowJo software (Tree Star).

Kauts M.L., De Leo B., Rodríguez-Seoane C., Ronn R., Glykofrydis F., Maglitto A., Kaimakis P., Basi M., Taylor H., Forrester L., Wilkinson A.C., Göttgens B., Saunders P, & Dzierzak E. (2018). Rapid Mast Cell Generation from Gata2 Reporter Pluripotent Stem Cells. Stem Cell Reports, 11(4), 1009-1020.

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Tumor Immune Cell Profiling

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Tumors were harvested, weighed, and processed to obtain single-cell suspensions. In brief, tumor tissues were dissociated and digested with 250 µg/mL of Liberase (Roche) and incubated for 30 min at 37 °C, while shaking at 105 rpm for proper digestion. Fetal bovine serum was then added to stop the reaction, and samples were filtered and washed with PBS + 2% FBS. The cell count per sample was performed, then samples were stained using fluorochrome-conjugated antibodies from BioLegend including: CD45 Pacific blue, cat# 103126; CD4 BV605, cat# 100451; CD8 PE, cat# 100707; CD49b APC, cat# 108910; Foxp3 Alexa488, cat# 126406; TIGIT PE-Cy7, cat# 142108; Gr1 BV510, cat# 108437; CD11b APC-fire750, cat# 101262; F4/80 Alexa700, cat# 123130; CD206 PercpCy5.5, cat# 141716. In alternate panels, a set of other antibodies was also used including: CD4 FITC, cat# 100406; CD8 PErcpCy5.5, cat# 100734; Gr1 APC, cat# 108412; CD11c BV510, cat# 117337; and PVR (CD155) PE, cat# 132206. Samples were run on the Attune flow cytometer, and data were analyzed using Flow-Jo 10 software.

Barsoumian H.B., Sezen D., Menon H., Younes A.I., Hu Y., He K., Puebla-Osorio N., Wasley M., Hsu E., Patel R.R., Yang L., Cortez M.A, & Welsh J.W. (2022). High Plus Low Dose Radiation Strategy in Combination with TIGIT and PD1 Blockade to Promote Systemic Antitumor Responses. Cancers, 14(1), 221.

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Cytotoxicity and Degranulation Assay for NK Cells

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NK cells were isolated by negative selection (Stem cell, 19855) from Il27ra^−/− and Il27ra^+/− mice and incubated with lymphoblast target cell line YAC-1(RRID: CVCL_2244) in 1:1 ratio at 10,000 per well of 96-well plate in complete RPMI media for 90 min at 37 C. Supernatant was used to assay cytotoxic efficiency of NK cells using CytoTox cell-mediated cytotoxicity assay (Promega, G1780) according to manufacturer’s protocol. Absorbance of light was measured at 490 nm. Killing efficiency was calculated as a ratio of experimental values to positive control (lysed target cells).
For degranulation assay, NK cells after the incubation with YAC1 were stained on ice for 30 min with antibodies cocktail: NK1.1-FITC (PK136; BioLegend, 108706), CD49a-Cy7/PE (HMα1; BioLegend, 142608), CD49b-APC (DX5, BioLegend, 108910), CD45-PerCP (30F-11; BioLegend, 103130), TCRβ-Alexa Fluor 700 (H57–597; BioLegend, 109224) and CD107a-PE, [1D4B; BioLegend, 121612, RRID:AB_2134487) and analyzed on BD Aria II flow cytometer. LIVE/DEAD Fixable Yellow Dead Cell stain (Invitrogen, L34959) was used to exclude dead cells. CD107a was used as a marker of degranulation.

Aghayev T., Mazitova A.M., Fang J.R., Peshkova I.O., Rausch M., Hung M., White K.F., Masia R., Titerina E.K., Fatkhullina A.R., Cousineau I., Turcotte S., Zhigarev D., Marchenko A., Khoziainova S., Makhov P., Tan Y.F., Kossenkov A.V., Wiest D.L., Stagg J., Wang X.W., Campbell K.S., Dzutsev A.K., Trinchieri G., Hill J.A., Grivennikov S.I, & Koltsova E.K. (2022). Interleukin-27 signaling serves as an immunological checkpoint for innate cytotoxic cells to promote hepatocellular carcinoma. Cancer discovery, 12(8), 1960-1983.

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Multiparameter Flow Cytometry of Immune Cells

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Single cell suspensions were stained with monoclonal antibodies for 1 hour at 4°C. One to four‐color flow cytometry was performed (FACS LSR, Becton Dickinson). Data were acquired using the CellQuest software (Becton Dickinson) using at least 100 000 events gated for live cells.
The following conjugated rat anti‐mouse mAbs were used: CD45‐PerCP, CD11b‐APC, CCR4‐PE, CCR5‐PE, CCR7‐PE, CCR10‐PE and CXCR4‐PE (BD Biosciences); CD49b‐APC (Biolegend); CD226‐PE, CD4‐FITC, CD25‐APC, CD8a‐FITC, PD1‐PE, CTLA4‐PE, PDL1‐PE‐Cy7, PDL2‐FITC, CD47‐APC (eBiosciences), CD11c‐FITC, GR1‐FITC (Miltenyi Biotech), CD206 (Santa Cruz), and anti‐mouse Foxp3 staining set PE (eBioscience).

El Hafny‐Rahbi B., Brodaczewska K., Collet G., Majewska A., Klimkiewicz K., Delalande A., Grillon C, & Kieda C. (2021). Tumour angiogenesis normalized by myo‐inositol trispyrophosphate alleviates hypoxia in the microenvironment and promotes antitumor immune response. Journal of Cellular and Molecular Medicine, 25(7), 3284-3299.

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Multiparametric Flow Cytometry Analysis

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Cells were collected and washed with FACS buffer (3% HI-FBS and 0.02% sodium azide in PBS). Then, single-cell suspensions were stained with anti-mouse CD3 (PerCP/Cy5.5, BioLegend, USA), CD49b (APC, BioLegend), CD69 (FITC, eBioscience), or NKG2D/CD314 (PE, BioLegend) antibodies in FACS buffer and incubated for 30 min on ice in the dark, followed by flow cytometry analysis. For intracellular cytokine staining, cells were restimulated with PMA/ionomycin (eBioscience) and brefeldin A (eBioscience) and incubated at 37℃ for 4 h. After cell surface staining, cells were incubated with IC Fixation Buffer (eBioscience) for 30 min and washed using 1X Permeabilization Buffer (eBioscience). Cells were stained with anti-mouse granzyme B (FITC, eBioscience), perforin (PE, eBioscience), or IFN-γ (FITC, BioLegend) antibodies in 1X Permeabilization Buffer for 30 min. Cells were resuspended in FACS buffer, and the results were detected and analyzed by flow cytometry.

Oh E.Y., & Lee S. (2021). Natural Killer Cell Activation by Weissella cibaria JW15 Isolated from Kimchi. Journal of Bacteriology and Virology, 51(2), 62-73.

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