Alexa fluor 488 conjugated cleaved caspase 3 asp175 d3e9 antibody

Manufactured by Cell Signaling Technology

The Alexa Fluor 488 conjugated cleaved caspase-3 (Asp175) (D3E9) antibody is a fluorescently labeled antibody that specifically recognizes the cleaved form of caspase-3 protein. Caspase-3 is a key effector of apoptosis, and this antibody can be used to detect and visualize apoptotic cells.

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2 protocols using alexa fluor 488 conjugated cleaved caspase 3 asp175 d3e9 antibody

Flavonoids and Doxorubicin Cytotoxicity Evaluation

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Apigenin, Apigenin-7-O-glucoside, naringenin and doxorubicin were purchased from Sigma (St. Louis, MO). Flavonoids were dissolved in sterile dimethyl sulfoxide (DMSO, Sigma) and doxorubicin was dissolved in sterile H₂O. Growth factor reduced basement membrane Matrigel was obtained from Corning (Bedford, MA). insulin-transferrin-selenium-X (ITS-X) and penicillin–streptomycin-glutamine solutions (PSG) were from GIBCO (Waltham, MA). Epidermal growth factor (EGF), hydrocortisone, insulin and gentamycin were purchased from Sigma. Alexa Fluor 488 conjugated cleaved caspase-3 (Asp175) (D3E9) antibody was purchased from Cell Signaling Technologies (Danvers, MA). Anti-phospho-histone γH2AX (S139, clone JBW301) antibody was from Millipore (Billerica, MA). Anti-hnRNPA2/B1 (clone DP3B3) and anti-β-tubulin (clone AA2) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Caspase-3 inhibitor DEVD-FMK and the caspase tetrapeptide substrates LEHD-, IETD- and DEVD-AFC were from Enzyme System Product (Livermore, CA).

Sudhakaran M., Parra M.R., Stoub H., Gallo K.A, & Doseff A.I. (2020). Apigenin by targeting hnRNPA2 sensitizes triple-negative breast cancer spheroids to doxorubicin-induced apoptosis and regulates expression of ABCC4 and ABCG2 drug efflux transporters. Biochemical pharmacology, 182, 114259.

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Apoptosis and Stemness Markers Analysis

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Cell apoptosis was analyzed using an Annexin V-FITC Apoptosis Detection kit (BD, bioscience) according to the manufacturer’s instruction. Briefly, cells were trypsinized and seeded in 60-mm culture dish. After treatment with cisplatin (10 μg/ml) for 12 h or docetaxel (0.01 μg/ml) for 24 h, cells were harvested, washed twice with PBS and stained with Annexin V-FITC and propidium iodide (PI) for 15 min.
Cleaved caspase-3 were analyzed using Alexa Fluor® 488-conjugated cleaved caspase-3 (Asp175) (D3E9) antibody (1:500, #9603, Cell Signaling Technology) with Alexa Fluor® 488-conjugated rabbit (DA1E) mAb IgG XP® Isotype antibody (#2975, Cell Signaling Technology) as a reference control. CD133 was analyzed by PE-conjugated anti-human CD133 antibody (1:50, 372,804, BioLegend) with PE-conjugated Mouse IgG1, κ isotype ctrl (FC) antibody (400,114, BioLegend) as a reference control.
Each assay had been performed for three times.

Wu Z., Liu Z., Jiang X., Mi Z., Meng M., Wang H., Zhao J., Zheng B, & Yuan Z. (2019). Depleting PTOV1 sensitizes non-small cell lung cancer cells to chemotherapy through attenuating cancer stem cell traits. Journal of Experimental & Clinical Cancer Research : CR, 38, 341.

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