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A Multiplex Assay to Simultaneously Monitor Apoptosis and Necrosis Using the Cellaca® PLX Image Cytometer

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Abstract

Apoptosis is the programmed cell death pathway that is critical for maintaining homeostasis, in which cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare different cancer therapeutics (i.e., small molecules, antibody drugs, cell therapies) that can initiate the process of apoptosis, enabling the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V, Caspase-3, and Propidium Iodide (PI) using the Cellaca® PLX Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA). First, apoptosis was induced in Jurkat and K562 cell lines with staurosporine over the course of 24 h, where apoptosis and necrosis were assessed at 0, 1, 1.5, 2, 4, 20, and 24 h timepoints. Samples were stained with Hoechst 33342 (total dye), Annexin V-APC (early-stage apoptosis), Caspase-3 488 (late-stage apoptosis), and PI (necrosis) at each timepoint and evaluated using image cytometry. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early- to late-stage apoptosis, and ultimately necrosis. A clear trend was observed analyzing apoptotic and necrotic populations during the first 1.5 h, showing differences of up to ~15% in single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis between apoptotic populations only, Annexin V+ only populations were higher than Caspase-3+ only populations by up to ~20% between 0 and 1.5 h. Conversely, K562 cells did not exhibit a notable change in apoptotic and necrotic populations due to low sensitivity to staurosporine. The proposed image cytometric detection method may provide an effective and efficient tool for rapid and reliable simultaneous detection of early- late-stage apoptosis, and necrosis. Therefore, allowing researchers to better characterize and screen potential cancer therapeutic drug candidates for their treatment efficacy in a higher throughput manner.

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Data is provided within the manuscript or supplementary information files.

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Authors and Affiliations

  1. Department of Advanced Technology R&D, Revvity Health Sciences, Inc., 360 Merrimack St., Suite 200, Lawrence, MA, 01843, USA

    Mackenzie Pierce, Yongyang Huang, Allen Lin, Carolina Franco Nitta, Dmitry Kuksin, Bo Lin & Leo Li-Ying Chan

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  1. Mackenzie Pierce
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  2. Yongyang Huang
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  3. Allen Lin
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  4. Carolina Franco Nitta
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  5. Dmitry Kuksin
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  6. Bo Lin
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  7. Leo Li-Ying Chan
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Contributions

Mackenzie Pierce, Yongyang Huang, Allen Lin, Carolina Franco Nitta, Dmitry Kuksin, Bo Lin, and Leo Li-Ying Chan contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mackenzie Pierce, Yongyang Huang, Allen Lin, Carolina Franco Nitta, Dmitry Kuksin, Bo Lin, and Leo Li-Ying Chan. The first draft of the manuscript was written by Mackenzie Pierce and all authors commented on subsequent versions of the manuscript. All authors read and approved the final manuscript. All trademarks, service marks, trade names, and brand names are the property of their respective owners.

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Correspondence to Leo Li-Ying Chan.

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Conflict of interest

The authors MP, YH, AL, CFN, DK, BL, and LLC declare competing financial interests. The work performed in the manuscript is for reporting on applications of an instrument from Revvity Health Sciences, Inc. (an indirect parent company of Nexcelom Biosciences, LLC.). The performed experiment is to demonstrate a multiplex fluorescence imaging methods for identifying early- and late-stage apoptosis.

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Electronic Supplementary Material

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10895_2024_3590_MOESM1_ESM.xlsx

Supplementary Material 1: Table 1 Staurosporine-treated Jurkat cell population percentage identification of Annexin V with respect to PI, Caspase-3 with respect to PI, and Annexin V with respect to Caspase-3. Cells showed an increase in apoptotic and necrotic populations as time in treatment advanced.

10895_2024_3590_MOESM2_ESM.xlsx

Supplementary Material 2: Table 2 Untreated control Jurkat cell population percentage identification of Annexin V with respect to PI, Caspase-3 with respect to PI, and Annexin V with respect to Caspase-3. Cells showed low levels of apoptotic and necrotic populations as time advanced.

10895_2024_3590_MOESM3_ESM.xlsx

Supplementary Material 3: Table 3 Staurosporine-treated K562 cell population percentage identification of Annexin V with respect to PI, Caspase-3 with respect to PI, and Annexin V with respect to Caspase-3. Cells showed low levels of apoptotic and necrotic populations as time in treatment advanced.

10895_2024_3590_MOESM4_ESM.xlsx

Supplementary Material 4: Table 4 Untreated control K562 cell population percentage identification of Annexin V with respect to PI, Caspase-3 with respect to PI, and Annexin V with respect to Caspase-3. Cells showed low levels of apoptotic and necrotic populations as time advanced.

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Pierce, M., Huang, Y., Lin, A. et al. A Multiplex Assay to Simultaneously Monitor Apoptosis and Necrosis Using the Cellaca® PLX Image Cytometer. J Fluoresc (2024). https://doi.org/10.1007/s10895-024-03590-3

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