CytoFlex Flow Cytometer Application Notes

Serum starvation of U937 cells increased the fluorescent signal in the FITC channel by one half log, while only a modest increase was seen for OCI/AML3 cells (Figure 2; Column 3). This indicates that the proportion of autophagic vesicles in U937 cultured cells increased following serum starvation, while the proportion of autophagic vesicles in OCI/AML3 cells remained relatively constant; every cell line responds to serum starvation differently, as demonstrated here. While the ‘control’ OCI/AML3 cells demonstrated lower overall viability than the ‘control’ U937 cells, viability was more closely correlated to increased autophagy in response to starvation, as described in the literature. 4 The presence of autophagic vesicles was measured by fluorescence in the FITC channel by the CytoFLEX Flow Cytometer. Cyto-ID is effective for the detection of autophagy, as it results in only minimal lysosomal staining. Using the above gating strategy (Figure 2), one can reliably identify cells containing autophagosomes from cells without autophagosomes, based on cell health and autophagic staining. Tips for success • Carefully read cell-thawing specifications prior to experimentation and ensure that culture growth phase is controlled in the experimental conditions. • Keep the Cyto-ID reagent away from light; carry out all Cyto-ID incubation steps in the dark Conclusions In summary, it is possible to identify cells containing autophagosomes using Cyto-ID Autophagy Detection Kit and the CytoFLEX Flow Cytometer. The protocol for rapid detection of Cyto-ID-stained autophagic vesicles in serum-starved and control leukemia cells could easily be extended to other cell lines and cell types. Using this cost-effective, compact laboratory solution, researchers are able to identify the graded and variable appearance of autophagosomes in cell culture. Notes The results shown here represent data generated on the Beckman Coulter CytoFLEX Flow Cytometer. Due to differences in the performance of makes and models of flow cytometers, the authors cannot guarantee similar results with the use of other flow cytometers. CytoFLEX is for Research Use Only (RUO), not intended or validated for use in the diagnosis of disease or other conditions. References 1. Mariño, G., Niso-Santano, M., Baehrecke, E.H., and Kroemer, G., Self-consumption: the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol. (2014) 15:81-94.

2. Liang, X.H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H., and Levine, B., Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. (1999) 402:672-6.

3. Mathew, R., Kongara, S., Beaudoin, B., Karp, C.M., Bray, K., Degenhardt, K., Chen, G., Jin, S., and White, E., Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev. (2007) 21:1367-81.

4. Chen, N., and Debnath, J., Autophagy and tumorigenesis. FEBS Lett. (2010) 584:1427-1435.

5. Ogier-Denis, E., and Codogno, P., Autophagy: a barrier or an adaptive response to cancer? Biochim. Biophys Acta. (2003) 1603:113–128.

6. Tanida, I., Ueno, T., and Kominami, E., LC3 and autophagy. Autophag Phag. (2008) 445:77-88.

For Research Use Only. Not for use in diagnostic procedures. Cyto-ID is a registered trade mark of ENZO Life Sciences

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