Abstract
Flow cytometry is an advanced technology for efficient, rapid, specific and multi-parameter analysis of single cells in various basic research fields including cytobiology, immunology, genetic, hematology and other basic research. Beclin-1 protein is an important indicator in monitoring autophagic activity. However, quantitative flow cytometry had been rarely reported till now to be applied in the detection of Beclin-1 expression. The present study was aimed to establish a flow cytometric method for quantitative detection of Beclin-1 expression by employing the autophagy inhibitor 3-methyladenine as the control. A multi-parameter optimal method for Beclin-1 protein staining is as follows. 2 % bovine serum albumin in phosphate buffered saline was used for sample block. Concentration of primary antibody was 0.004 μg/μL. Samples were incubated at room temperature (25 °C) for 30 min. The prepared samples had better to be detected immediately or to be stored at 4 °C and detected within 6 h, otherwise the samples should be fixed in 1 % paraformaldehyde storing at 4 °C and detected within 3 d. Furthermore, we employed the immunohistochemistry to validate the method in vivo, the results confirmed flow cytometric method. The established flow cytometric analysis for Beclin-1 protein has the advantage of simpleness, speediness, sensitivity and reproducibility.
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Acknowledgments
This work was supported by the Guangdong Natural Science Foundation of China (No. 2003C34403). We would like to express our sincere thanks to the reviewers and editors for the constructive and positive comments.
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Yuping He, Zhentao Mo, and Zhongfeng Xue contributed equally to this work.
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He, Y., Mo, Z., Xue, Z. et al. Establish a flow cytometric method for quantitative detection of Beclin-1 expression. Cytotechnology 65, 481–489 (2013). https://doi.org/10.1007/s10616-012-9503-9
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DOI: https://doi.org/10.1007/s10616-012-9503-9