PMID- 38294633
OWN - NLM
STAT- Publisher
LR  - 20240131
IS  - 1573-4994 (Electronic)
IS  - 1053-0509 (Linking)
DP  - 2024 Jan 31
TI  - A Multiplex Assay to Simultaneously Monitor Apoptosis and Necrosis Using the 
      Cellaca(R) PLX Image Cytometer.
LID - 10.1007/s10895-024-03590-3 [doi]
AB  - 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(R) 
      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.
CI  - (c) 2024. The Author(s), under exclusive licence to Springer Science+Business 
      Media, LLC, part of Springer Nature.
FAU - Pierce, Mackenzie
AU  - Pierce M
AD  - Department of Advanced Technology R&D, Revvity Health Sciences, Inc., 360 
      Merrimack St., Suite 200, Lawrence, MA, 01843, USA.
FAU - Huang, Yongyang
AU  - Huang Y
AD  - Department of Advanced Technology R&D, Revvity Health Sciences, Inc., 360 
      Merrimack St., Suite 200, Lawrence, MA, 01843, USA.
FAU - Lin, Allen
AU  - Lin A
AD  - Department of Advanced Technology R&D, Revvity Health Sciences, Inc., 360 
      Merrimack St., Suite 200, Lawrence, MA, 01843, USA.
FAU - Franco Nitta, Carolina
AU  - Franco Nitta C
AD  - Department of Advanced Technology R&D, Revvity Health Sciences, Inc., 360 
      Merrimack St., Suite 200, Lawrence, MA, 01843, USA.
FAU - Kuksin, Dmitry
AU  - Kuksin D
AD  - Department of Advanced Technology R&D, Revvity Health Sciences, Inc., 360 
      Merrimack St., Suite 200, Lawrence, MA, 01843, USA.
FAU - Lin, Bo
AU  - Lin B
AD  - Department of Advanced Technology R&D, Revvity Health Sciences, Inc., 360 
      Merrimack St., Suite 200, Lawrence, MA, 01843, USA.
FAU - Chan, Leo Li-Ying
AU  - Chan LL
AD  - Department of Advanced Technology R&D, Revvity Health Sciences, Inc., 360 
      Merrimack St., Suite 200, Lawrence, MA, 01843, USA. leo.chan@revvity.com.
LA  - eng
PT  - Journal Article
DEP - 20240131
PL  - Netherlands
TA  - J Fluoresc
JT  - Journal of fluorescence
JID - 9201341
SB  - IM
OTO - NOTNLM
OT  - Annexin
OT  - Apoptosis
OT  - Caspase
OT  - Cellaca(R) PLX
OT  - Image cytometry
OT  - Necrosis
EDAT- 2024/01/31 12:42
MHDA- 2024/01/31 12:42
CRDT- 2024/01/31 11:15
PHST- 2023/12/11 00:00 [received]
PHST- 2024/01/15 00:00 [accepted]
PHST- 2024/01/31 12:42 [medline]
PHST- 2024/01/31 12:42 [pubmed]
PHST- 2024/01/31 11:15 [entrez]
AID - 10.1007/s10895-024-03590-3 [pii]
AID - 10.1007/s10895-024-03590-3 [doi]
PST - aheadofprint
SO  - J Fluoresc. 2024 Jan 31. doi: 10.1007/s10895-024-03590-3.