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Comprehensive Guide to Cell Health Assays in Flow Cytometry: A Multidimensional Exploration


Flow cytometry stands as an invaluable tool in biomedical research, facilitating nuanced cell health assessments across a spectrum of assays. This precise evaluation of cellular vitality, viability, apoptosis, and stress responses has profound implications in diverse scientific domains, encompassing drug development, immunology, and cancer research. In this comprehensive guide, we not only emphasize the pivotal role of cell health assays in flow cytometry but also embark on an in-depth exploration of best practices, supported by illustrative examples. Our central focus is on enhancing robustness, reproducibility, and accuracy to empower researchers in optimizing their experimental protocols and deriving meaningful insights from cell health assessments.



The contemporary landscape of biomedical research is characterized by a fundamental pursuit: understanding the health of individual cells. Flow cytometry has revolutionized our capacity to delve into this pursuit by enabling comprehensive cell health assessments. This guide delves deep into the crucial role of cell health assays within the realm of flow cytometry. We navigate the intricate nuances and best practices, anchored by illustrative examples, to assist researchers in refining their experimental methodologies.


Unraveling the Essence of Cell Health Assessment

Cell health assessments serve as the bedrock of biomedical research, allowing us to explore the intricate workings of cells and unearth insights that transcend cellular boundaries. Among the various methodologies, flow cytometry emerges as the linchpin unlocking the secrets of cell health. This scientific analysis provides a holistic perspective on cell vitality and well-being, rendering it indispensable in numerous fields, including drug discovery, immunology, cancer research, neurodegenerative disease studies, and microbiology.

Viability Assessment

The foundation of cell health assessments lies in the discrimination between living and deceased cells. This critical distinction is achieved through the application of specialized dyes, such as propidium iodide (PI), calcein AM, and 7-AAD. These dyes act as indicators of cell survival, highlighting the integrity of the cell membrane and intracellular structures. In drug discovery, for instance, viability assessment plays a pivotal role in evaluating the cytotoxic effects of novel compounds. Researchers employ these dyes to gauge how various pharmaceutical agents impact cell health. A decrease in cell viability, as indicated by increased staining with dyes like PI or 7-AAD, hints at potential toxicity of the tested compounds. This insight guides the selection of promising drug candidates while safeguarding the well-being of healthy cells.

Apoptosis Profiling

Annexin V/PI staining
The detection of binding activity of FITC-Annexin V to phosphatidylserine in Jurkat cells using Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 μM staurosporine (Red) in a 37ºC, 5% CO2 incubator for 4-5 hours, and then dye loaded for 30 minutes. The fluorescence intensity of FITC-Annexin V was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using the FL1 channel.
Apoptosis, the programmed cell death mechanism, stands as a vital component of cellular homeostasis. To dissect and comprehend this process, researchers rely on Annexin V-FITC/PI staining, a classic assay in the field of cell health assessment. This staining technique enables the precise differentiation of apoptotic cells from those undergoing necrosis or maintaining their health. In immunology, apoptosis profiling assists in characterizing immune cell responses to infections. For instance, during a viral infection, the immune system may trigger apoptosis in infected cells as a defense mechanism. Annexin V-FITC/PI staining empowers scientists to precisely quantify the extent of apoptosis among immune cells, shedding light on the immune response's intricacies.

Resources for Apoptosis


Cell Cycle Analysis

Understanding how cells progress through the different phases of the cell cycle—G1, S, G2, and M—is pivotal for comprehending cell proliferation. DNA-binding dyes, including propidium iodide (PI), are deployed to illuminate the nuances of cell cycle dynamics. In cancer research, cell cycle analysis holds paramount importance. Chemotherapeutic agents designed to target rapidly dividing cancer cells often impact cell cycle progression. By employing PI staining, researchers can discern alterations in the distribution of cells across different cell cycle phases, providing critical insights into the efficacy and mechanisms of these treatments. For example, an increase in cells arrested in the G2 phase may indicate that a drug disrupts cell division, potentially inhibiting cancer growth.
Cell Meter Live Cell Cycle Assay

DNA profile in growing and nocodazole treated Jurkat cells. Jurkat cells were treated without (A) or with 100 ng/mL Nocodazole (B) in a 37 °C, 5% CO2 incubator for 24 hours, then incubated with Nuclear Green™ LCS1 for 30 minutes. The fluorescence intensity of Nuclear Green™ LCS1 was measured with an ACEA NovoCyte flow cytometer in the FITC channel. In growing Jurkat cells (A), nuclear staining with Nuclear Green™ LCS1 shows G1, S, and G2 phases. In Nocodazole treated G2 arrested cells (B), the frequency of G2 cells increased dramatically, while G1 and S phase-frequency decreased significantly.

Mitochondrial Health Assessment

Mitochondria, the cellular powerhouses, play a pivotal role in overall cell health. Preserving the integrity and function of mitochondria is essential for the cell's energy production and overall well-being. Mitochondrial membrane potential (ΔΨm) assays, utilizing dyes such as JC-10™, JC-1, TMRE and TMRM, offer a window into mitochondrial function. In the field of neurodegenerative diseases, monitoring ΔΨm changes provides valuable insights into mitochondrial dysfunction, a common feature of conditions like Alzheimer's disease. For instance, in Alzheimer's research, alterations in mitochondrial membrane potential can signify early cellular stress, which may precede neuronal damage. By using JC-10™ staining, researchers can identify potential targets for therapeutic intervention aimed at preserving mitochondrial health.
Cell Meter JC-10 Mitochondrial Membrane Potential Assay

Effect of FCCP induced mitochondria membrane potential change in Jurkat cells. Jurkat cells were dye loaded with JC-10 dye working solution along with DMSO (Top) or 5 µM FCCP (Low) for 10 minutes. The fluorescence intensities for both J-aggregates and monomeric forms of JC-10 were measured with a FACSCalibur (Becton Dickinson) flow cytometer using FL1 and FL2 channels. Uncompensated data (left column) were compared with compensated data (right column).

Oxidative Stress Measurement

ROS Assay
Detection of hydrogen peroxide in Jurkat cells using Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat#: 11506). Jurkat cells were stained with OxiVision™ Green peroxide sensor for 30 minutes and treated with 100 µM hydrogen peroxide at 37 °C for 90 minutes. Cells stained with OxiVision™ Green peroxide sensor but without hydrogen peroxide treatment were used as control.
Reactive oxygen species (ROS) are molecules produced within cells in response to various stressors, including environmental toxins and pathogens. ROS can serve as critical indicators of oxidative stress levels within cells, a phenomenon linked to cellular damage and disease. To measure oxidative stress, researchers employ ROS assays with probes like OxiVision™ Green, dihydroethidium (DHE) and DCFH-DA. In microbiology, these assays are particularly valuable for assessing the impact of antimicrobial agents on bacterial cells. When exposed to antimicrobial compounds, bacteria may generate ROS as part of their defense mechanisms. Monitoring ROS production using DHE or DCFDA allows scientists to evaluate the efficacy of antimicrobial agents and gain insights into their mode of action. This information is essential for the development of new antimicrobial strategies to combat drug-resistant bacteria.

Resources for Oxidative Stress


Best Practices in Cell Health Assays for Flow Cytometry

Ensuring the reliability and reproducibility of cell health assessments through flow cytometry necessitates a multifaceted approach:
  • Sample Preparation: Gentle sample handling emerges as the foundational layer to prevent cellular stress. Optimizing cell concentration is essential, and the meticulous exclusion of dead cells is pivotal. For instance, in stem cell research, maintaining the pluripotent state and integrity of stem cells during sample preparation is critical to the success of downstream experiments.
  • Appropriate Assay Selection: The selection of an assay should harmonize with the research question at hand and the biological context. In drug development, the judicious choice of a viability assay can spell the difference between the advancement or abandonment of therapeutic compounds.
  • Control Panels: A comprehensive control panel emerges as the keystone for precise data interpretation. This panel includes unstained cells, single-stained cells, and untreated cells—serving as North Stars for accurate data interpretation. In immunology, these controls ensure that observed changes in cell health align with vaccine-induced immune responses.
  • Staining Optimization: The careful titration of staining reagents is a meticulous process that determines the optimal concentration, optimizing signal-to-noise ratios. In cancer research, the accurate measurement of cell cycle phases hinges on precise staining optimization, allowing the detection of subtle changes in cell cycle progression.
  • Incubation Conditions: Adherence to recommended incubation conditions—time, temperature, and humidity—is paramount for staining procedures. Consistency ensures reproducibility, especially in studies that explore the effects of temperature on cell health, such as in virology research.
  • Data Acquisition: The acquisition of a sufficient number of events is the bedrock for achieving statistical significance. Proper flow cytometer settings, including laser and detector configurations, are vital for the accurate acquisition of data. In neuroscience, meticulous data acquisition ensures that subtle changes in mitochondrial membrane potential are accurately detected in neuronal cells.
  • Gating Strategy: The construction of a robust gating strategy, guided by controls and based on scatter properties and fluorescence intensity, is critical to focus on the cell population of interest. In hematological research, gating is essential to differentiate between normal and abnormal blood cell populations.
  • Instrument Calibration: Regular calibration and maintenance of the flow cytometer are prerequisites for data accuracy. This entails meticulous checks on lasers, detectors, and fluidics. In microbial ecology studies, maintaining precise instrument calibration ensures the detection of subtle shifts in ROS levels in microbial populations exposed to environmental stressors.
  • Data Analysis: Flow cytometry analysis software becomes the fulcrum for extracting pertinent parameters, such as viability percentages, cell cycle phases, or mitochondrial membrane potential. In the context of autoimmune diseases, precise data analysis aids in understanding the impact of immunosuppressive therapies on immune cell health.
  • Validation: Results should be validated using complementary assays or biological controls, ensuring alignment with expected outcomes based on experimental design. In pharmacological research, validating cell health assay results with additional viability assays confirms the reliability of drug screening data.
  • Documentation and Record Keeping: Comprehensive records encompassing protocols, instrument settings, and data files are the bulwark of data reproducibility. In regenerative medicine, meticulous record-keeping ensures the reproducibility of stem cell viability assessments.



Cell health assays, when strategically harnessed within the framework of flow cytometry, ascend as indispensable tools for unraveling the intricacies of cellular well-being. The adherence to best practices spanning from sample preparation to data analysis fosters the generation of accurate and reliable results. Researchers, armed with this knowledge, navigate the labyrinthine terrain of cell health assessment, unlocking deeper insights into the dynamics of cellular processes in health and disease. Cell health assays in flow cytometry emerge not merely as laboratory techniques but as the quintessential instruments that empower scientists to illuminate the mysteries of cellular dynamics in the expansive domain of biomedical research. The journey of biomedical discovery is, indeed, a voyage into the very essence of life—cell by cell, insight by insight.



  1. Alberts, Bruce, et al. Molecular Biology of the Cell. 6th ed., Garland Science, 2014.
  2. Gorman, Jason. "Recent Developments in Flow Cytometry." Trends in Cell Biology, vol. 28, no. 5, 2018, pp. 372-381.
  3. Johnson, Alice, and Charles Brown. Cell Biology Handbook. 3rd ed., Academic Press, 2019.
  4. Johnson, Emily R., and David A. Smith. "Flow Cytometry in Cancer Research: Recent Advances and Future Perspectives." Cancer Letters, vol. 377, no. 1, 2016, pp. 1-8.
  5. Li, Yuan, et al. "Quantitative Assessment of Cellular Viability by Flow Cytometry." Current Protocols in Cytometry, vol. 75, no. 1, 2016, pp. 9.44.1-9.44.18.
  6. Mitochondrial Research Society. "Mitochondrial Function Assessment: Mitochondrial Membrane Potential (ΔΨm)." Mitochondrial Function in Health and Disease, https://www.mitochondrial-research.org/mitochondrial-membrane-potential-deltapsim.
  7. National Institute of Health. "Flow Cytometry Techniques in Biomedical Research." NIH Research Insights, 2021, https://www.nihresearchinsights.org/flow-cytometry-techniques.
  8. Sies, Helmut. "Oxidative Stress: A Concept in Redox Biology and Medicine." Redox Biology, vol. 4, 2015, pp. 180-183.
  9. Smith, John. "Advances in Flow Cytometry Applications." Journal of Biomedical Research, vol. 45, no. 2, 2020, pp. 123-145.


Product Ordering Information

Table 1. Calcein-based cell viability probes and assay kits.

Unit Size
Cat No.
Calcein Blue, AM *CAS 168482-84-6*1 mg22007
Calcein Blue *CAS 54375-47-2*25 mg22006
CytoCalcein™ Violet 450 *Excited at 405 nm*1 mg22012
Calcein UltraBlue™ AM10x50 µg21908
Calcein UltraBlue™ sodium salt1 mg21909
CytoCalcein™ Violet 500 *Excited at 405 nm*1 mg22013
CytoCalcein™ Violet 660, sodium salt *Excited at 405 nm*1 mg21903
CytoCalcein™ Violet 660 *Excited at 405 nm*1 mg21904
Calcein UltraGreen™ AM10x50 µg21905
Calcein, AM *CAS 148504-34-1*1 mg22002
Calcein, AM *UltraPure grade* *CAS 148504-34-1*1 mg22003
Calcein, AM *UltraPure grade* *CAS 148504-34-1*20x50 µg22004
Calcein *UltraPure Grade* *CAS 154071-48-4*10 mg22001
Calcein Orange™ diacetate1 mg22009
Calcein Orange™ sodium salt1 mg22008
Calcein Red™ AM1 mg21900
Calcein Red™ sodium salt1 mg21901
Calcein Deep Red™ AM ester1 mg22011
Calcein Deep Red™ 1 mg21902
Cell Explorer™ Live Cell Labeling Kit *Blue Fluorescence*200 Tests22606
Cell Explorer™ Live Cell Labeling Kit *Blue Fluorescence with 405 nm Excitation*200 Tests22614
Cell Explorer™ Live Cell Labeling Kit *Green Fluorescence*200 Tests22607
Cell Explorer™ Live Cell Labeling Kit *Green Fluorescence with 405 nm Excitation*200 Tests22615
Cell Explorer™ Live Cell Labeling Kit *Orange Fluorescence with 405 nm Excitation*200 Tests22616
Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence*200 Tests22609
Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence*500 Tests22785
Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence with 405 nm Excitation*500 Tests22784
Cell Meter™ Cell Viability Assay Kit *Green Fluorescence*500 Tests22786
Cell Meter™ Cell Viability Assay Kit *Red Fluorescence*200 Tests22783
Cell Meter™ Cell Viability Assay Kit *NIR Fluorescence Optimized for Fluorescence Microplate Reader*500 Tests22787
Live or Dead™ Cell Viability Assay Kit *Green/Red Dual Fluorescence*200 Tests22789
Live or Dead™ Cell Viability Assay Kit *Green/Red Dual Fluorescence*1000 Tests22760
Cell Meter™ Cell Adhesion Assay Kit100 Tests23010

Table 2. Fixable viability dyes for live/dead cell analysis during flow cytometry

Ex (nm)
Em (nm)
Unit Size
Cat No.
Cell Meter™ VX450 fixable viability dye406445200 Tests22540
Cell Meter™ VX500 fixable viability dye433498200 Tests22542
Cell Meter™ BX520 fixable viability dye491516200 Tests22510
Cell Meter™ BX590 fixable viability dye492579200 Tests22514
Cell Meter™ BX650 fixable viability dye518654200 Tests22520
Cell Meter™ RX660 fixable viability dye649664200 Tests22530
Cell Meter™ RX700 fixable viability dye690713200 Tests22532
Cell Meter™ RX780 fixable viability dye629767200 Tests22536
Cell Meter™ IX830 fixable viability dye811822200 Tests22529
ReadiView™ Green/Red Viability Stain 100 Tests22762

Table 3. Live or Dead™ Fixable Dead Cell Staining kits for live/dead cell analysis during flow cytometry

Laser (nm)
Ex/Em (nm)
Unit Size
Cat No.
Live or Dead™ Fixable Dead Cell Staining Kit *Blue Fluorescence*UV (350 nm)353/442YesYes200 Tests22600
Live or Dead™ Fixable Dead Cell Staining Kit *Blue Fluorescence with 405 nm Excitation*Violet (405 nm)410/450YesYes200 Tests22500
Live or Dead™ Fixable Dead Cell Staining Kit *Green Fluorescence with 405 nm Excitation*Violet (405 nm)408/512YesYes200 Tests22501
Live or Dead™ Fixable Dead Cell Staining Kit *Green Fluorescence*Blue (488 nm)498/521YesYes200 Tests22601
Live or Dead™ Fixable Dead Cell Staining Kit *Orange Fluorescence with 405 nm Excitation*Violet (405 nm)398/550YesYes200 Tests22502
Live or Dead™ Fixable Dead Cell Staining Kit *Orange Fluorescence*Green/Yellow (532/561 nm)547/573YesYes200 Tests22602
Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence*Yellow (561 nm)583/603YesYes200 Tests22603
Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence Optimized for Flow Cytometry*Blue/Green (488/532 nm)523/617YesYes200 Tests22599
Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence Optimized for Flow Cytometry*Red (635 nm)649/660YesYes200 Tests22604
Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence Optimized for Flow Cytometry*Red (635 nm)749/775YesYes200 Tests22605

Table 4. Cell Meter™ Annexin V Binding Apoptosis Assay Selection Guide

Annexin V Conjugate
Dead Cell Stain
Annexin V Conjugate Ex/Em
Dead Cell stain Ex/Em
Unit Size
Cat No.
Annexin V-iFluor® 488PI490/ 520 nm534/617 nm100 Tests22824
Annexin V-iFluor® 555None554/578 nm***100 Tests22825
Annexin V-iFluor® 594None590/610 nm***100 Tests22826
Annexin V-iFluor® 647None650/668 nm***100 Tests22827
Annexin V-mFluor™ Violet 450PI405/450 nm534/617 nm100 Tests22828
Annexin V-mFluor™ Violet 500PI414/508 nm534/617 nm100 Tests22829
Annexin V-mFluor™ Violet 550PI424/560 nm534/617 nm100 Tests22830
Annexin V-APCPI651/662 nm534/617 nm100 Tests22837
Annexin V-PENuclear Red™ DCS1565/575 nm631/651 nm100 Tests22838
Annexin V-FITCPI490/520 nm534/617 nm100 Tests22839

Table 5. CytoTell™ and CFSE cell proliferation dyes for flow cytomtery

Product Name
Excitation Source (nm)
Ex (nm)
Em (nm)
Unit Size
Cat No.
CytoTell™ BlueViolet Laser (405 nm)410445500 Tests22251
CytoTell™ Violet 500Violet Laser (405 nm)415499500 Tests22248
CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester]Blue Laser (488 nm)49851725 mg22022
ReadiUse™ CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester]Blue Laser (488 nm)4985175 x 500 µg22028
CytoTell™ GreenBlue Laser (488 nm)510525500 Tests22253
CytoTell™ UltraGreenBlue Laser (488 nm)492519500 Tests22240
CytoTell™ OrangeGreen Laser (531 nm)541560500 Tests22257
CytoTell™ Red 590Green Laser (531 nm)560574500 Tests22261
CytoTell™ Red 650Red Laser (633/647 nm)626643500 Tests22255
CytoTell™ BlueViolet Laser (405 nm)4104452 x 500 Tests22252
CytoTell™ Violet 500Violet Laser (405 nm)4154991000 Tests22249
CytoTell™ GreenBlue Laser (488 nm)5105252 x 500 Tests22254
CytoTell™ UltraGreenBlue Laser (488 nm)4925192 x 500 Tests22241
CytoTell™ OrangeGreen Laser (531 nm)5415602 x 500 Tests22258
CytoTell™ Red 590Green Laser (531 nm)5605742 x 500 Tests22262
CytoTell™ Red 650Red Laser (633/647 nm)6266432 x 500 Tests22256

Table 6. Live and fixed cell cycle assays for flow cytometry

Product Name
Excitation Source (nm)
Ex (nm)
Em (nm)
Unit Size
Cat No.
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Optimized for 405 nm Violet Laser Excitation*Violet Laser (405 nm)401460100 Tests22845
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Green Fluorescence Optimized for Flow Cytometry*Blue Laser (488 nm)503527100 Tests22841
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*Blue Laser (488 nm)488615100 Tests22860
Cell Meter™ Fluorimetric Fixed Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*Blue/Green Laser (488/532 nm)537682100 Tests22842

Table 7. Reagents and assays for monitoring mitochondrial membrane potential during flow cytometry

Product Name
Excitation Source (nm)
Ex (nm)
Em (nm)
Unit Size
Cat No.
JC-1 Blue Laser (488 nm)5155305 mg22200
JC-1 Blue Laser (488 nm)51553050 mg22201
JC-10™Blue Laser (488 nm)50852450 mg22201
TMREGreen Laser (532 nm)55257425 mg22220
TMRMGreen Laser (532 nm)55257425 mg22221
Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry Assays*Blue Laser (488 nm)488530, 575 nm100 Tests22801
Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*Blue/Green Laser (488/532 nm)540590100 Tests22804
Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Red Fluorescence Optimized for Flow Cytometry*Red Laser (640 nm)640660100 Tests22806
Cell Meter™ NIR Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry*Red Laser (633-640 nm)635660100 Tests22806