Apc conjugated anti cd44 antibody

Manufactured by BD

Sourced in United States

The APC-conjugated anti-CD44 antibody is a fluorescently-labeled monoclonal antibody that binds to the CD44 cell surface protein. CD44 is a cell adhesion molecule involved in various cellular processes. The antibody is conjugated to the fluorescent dye Allophycocyanin (APC), which allows for the detection and analysis of CD44-expressing cells using flow cytometry or other fluorescence-based techniques.

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Anti-CD44 (84 citations) CD44-APC (77 citations) Anti-CD44-APC (53 citations) APC-conjugated anti-CD44 (13 citations) APC-anti-CD44 antibody (9 citations) APC-conjugated CD44 (9 citations) Anti-CD44 antibody (6 citations) APC-conjugated CD44 antibody (5 citations) CD44 antibody (4 citations)

7 protocols using apc conjugated anti cd44 antibody

Identification of Cancer Stem Cells

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An APC-conjugated anti-CD44 antibody (BD Bioscience, clone G44-26), a PE-Cy7-conjugated anti-CD24 antibody (BioLegend, clone LM5), an APC-conjugated anti-ABCG2 antibody (clone 5D3, BD Bioscience) and propidium iodide (5 μg ml⁻¹; BD Bioscience) were used for fluorescence-activated cell sorting analyses. To detect the SP fraction, MCF7 and ZR75-1 cells were stained with 5 μg ml⁻¹ Hoechst 33342 (Invitrogen) in the presence or absence of 1 μM Ko143 (Sigma) at 37 °C for 90 min. Flow cytometric analysis and cell sorting were performed using a JSAN cell sorter (Bay bioscience) and the results were analysed with FlowJo software.

Takahashi R.U., Miyazaki H., Takeshita F., Yamamoto Y., Minoura K., Ono M., Kodaira M., Tamura K., Mori M, & Ochiya T. (2015). Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1. Nature Communications, 6, 7318.

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Identification of Breast Cancer Stem Cells

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Cells were trypsinized by dissociation buffer (Invitrogen) and resuspended in blocking solution (Ca²⁺, Mg²⁺-free HBSS containing 2% goat serum). Next, cells were incubated with APC-conjugated anti-CD44 antibody (BD Bioscience) and PE-conjugated anti-CD24 antibody (BD Bioscience) for 1 h at 4 °C. After washes, the labeled cells were analyzed by a Beckman CytoFLEX flow cytometer. The breast cancer stem cell population (CD44^high and CD24^low/−) was calculated via the CytExpert software.

Chen H.Y., Chan S.J., Liu X., Wei A.C., Jian R.I., Huang K.W., Lang Y.D., Shih J.H., Liao C.C., Luan C.L., Kao Y.T., Chiang S.Y., Hsiao P.W., Jou Y.S., Chen Y, & Chen R.H. (2022). Long noncoding RNA Smyca coactivates TGF-β/Smad and Myc pathways to drive tumor progression. Journal of Hematology & Oncology, 15, 85.

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Phenotypic Profiling of Cell Populations

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Cells were collected and washed twice with permeabilization wash buffer and were then resuspended in flow cytometry staining buffer (Bio-Rad), and cell numbers were counted. The dissociated cells were then stained with APC-conjugated anti-CD44 antibody (1:50, Cat# 559942, BD Pharmingen) and FITC-conjugated anti-CD24 antibody (1:50, Cat# 555427, BD Pharmingen) for 30 min at 4 °C. After washing twice in flow cytometry buffer, the cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences). For the ALDEFLUOR Assay, cells were harvested by trypsinization, washed in PBS, labeled with Aldefluor Reagent (StemCell Technologies, Grenoble, France) and incubated at 37 °C for 45 min. Finally, all samples were analyzed by a FACS machine (BD Calibur).

Xiao G., Lu W., Yuan J., Liu Z., Wang P, & Fan H. (2024). Fbxw7 suppresses carcinogenesis and stemness in triple-negative breast cancer through CHD4 degradation and Wnt/β-catenin pathway inhibition. Journal of Translational Medicine, 22, 99.

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CD24 and CD44 Expression Analysis

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For the analysis of CD24 and CD44 expression on cell membrane, 5 × 10⁵ of cells were collected in Cell Staining Buffer (BioLegend, #420201) and stained with PE-Cy™7-conjugated anti-CD24 (1:100 for 20 min; BD Biosciences, #561646) and APC-conjugated anti-CD44 antibody (1:60 for 20 min; BD Biosciences, #559942) by using PE-Cy™7 Mouse IgG2a (1:100 for 20 min; BD Biosciences, #552868) and APC Mouse IgG2b (1:60 for 20 min; BD Biosciences, #555745) as control staining. Stained cells were analyzed by BD FACSCanto II cytometer and data were acquired by BD FACSDiva v8.0.2 software and processed by FlowJo v10.7.1 software (BD Biosciences). ALDEFLUOR assay was carried out using the ALDEFLUOR assay kit (STEMCELL Inc., #101700) according to the manufacturer’s instructions. The ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB), was used as a negative control. The processed cells were evaluated by BD FACSCanto II cytometer and data were acquired by BD FACSDiva v8.0.2 software and analyzed by FlowJo v10.7.1 software (BD Biosciences).

Lee H.H., Wang Y.N., Yang W.H., Xia W., Wei Y., Chan L.C., Wang Y.H., Jiang Z., Xu S., Yao J., Qiu Y., Hsu Y.H., Hwang W.L., Yan M., Cha J.H., Hsu J.L., Shen J., Ye Y., Wu X., Hou M.F., Tseng L.M., Wang S.C., Pan M.R., Yang C.H., Wang Y.L., Yamaguchi H., Pang D., Hortobagyi G.N., Yu D, & Hung M.C. (2021). Human ribonuclease 1 serves as a secretory ligand of ephrin A4 receptor and induces breast tumor initiation. Nature Communications, 12, 2788.

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Dual Labeling of CD44 and CD24

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PTX-EXO and/or si-circBACH1 treated cells in the presence of PTX were dual-labeled with a APC-conjugated anti-CD44 antibody and an FITC-conjugated anti-CD24 antibody (BD Pharmingen, San Diego). 1 × 10⁵ cells were labeled and incubated with FITC-conjugated anti-CD24 and APC-conjugated anti-CD44 antibody at 4 °C in the dark for 1 h. The cells were washed and then analyzed by flow cytometer (BD FACS Aria II).

Xia W., Chen W., Ni C., Meng X., Wu J., Yang Q., Tang H., Yuan H, & Fang S. (2023). Chemotherapy-induced exosomal circBACH1 promotes breast cancer resistance and stemness via miR-217/G3BP2 signaling pathway. Breast Cancer Research : BCR, 25, 85.

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Flow Cytometry-Based CD44 and HA-CPN Sorting

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One million cells were stained with anti CD44 APC-conjugated antibody (BD, #559,942, 20 μl per 10⁶ cells in 100 µl of stain buffer) on ice for 45 min, washed and suspended in PBS/2 mM EDTA at the concentration of 10⁶ cells/mL. The cells were sorted using BD FACSAria Fusion™ cell sorter (BD) to separate CD44 + from CD44- cells and/or Ha-CPN + and HA-CPN- cells.

Lubanska D., Alrashed S., Mason G.T., Nadeem F., Awada A., DiPasquale M., Sorge A., Malik A., Kojic M., Soliman M.A., deCarvalho A.C., Shamisa A., Kulkarni S., Marquardt D., Porter L.A, & Rondeau-Gagné S. (2022). Impairing proliferation of glioblastoma multiforme with CD44+ selective conjugated polymer nanoparticles. Scientific Reports, 12, 12078.

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Identification of Mucoepidermoid Carcinoma CSCs

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UM-HMC-3A cells were resuspended and counted using a Countess II FL automatic cell counter (Invitrogen, Carlsbad, CA, USA). The combination of ALDH^bright plus CD44^high was used to identify mucoepidermoid carcinoma cancer stem cells as previously reported[27 (link)]. The Aldefluor kit (StemCell Technologies, Durham, NC, USA) was used according to the manufacturer’s instructions to identify cells with high ALDH1 enzymatic activity. UM-HMC-3A cells were suspended with activated Aldefluor substrate (BODIPY amino acetate) or negative control (dimethylamino benzaldehyde, a specific ALDH inhibitor) for up to 45 minutes at 37°C. Then, cells were washed and suspended with anti-CD44/APC conjugated antibody (BD Biosciences, Mountain View, CA, USA) and incubated for 25 min in shaking rotor at 4°C. All samples were analyzed using a flow cytometer Accuri C6 (BD Biosciences, USA) equipped with two excitation lasers: a solid blue state (488 nm) and a diode red (640nm). All assays were performed in sextuplicate. All CSC flow cytometry experiments for the various drugs (Erlotinib, SAHA and CUDC-101) were performed concurrently.

Parag-Sharma K., Tasoulas J., Musicant A.M., Viesi do Nascimento-Filho C.H., Zhu Z., Twomey C., Liu P., Castilho R.M, & Amelio A.L. (2021). Synergistic Efficacy of Combined EGFR and HDAC Inhibitors Overcomes Tolerance to EGFR Monotherapy in Salivary Mucoepidermoid Carcinoma. Oral oncology, 115, 105166.

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