Skip to main content

Advertisement

SpringerLink
Log in

The Detailed Comparison of Cell Death Detected by Annexin V-PI Counterstain Using Fluorescence Microscope, Flow Cytometry and Automated Cell Counter in Mammalian and Microalgae Cells

  • ORIGINAL ARTICLE
  • Published:
Journal of Fluorescence Aims and scope Submit manuscript

Abstract

The evaluation of cell wellness is an important task for molecular biology research. This mainly comprises the assessment for morphology and viability of culturing cells. Annexin V-Propidium iodide counterstaining has been currently one of the common and easy methods to discriminate apoptotic and necrotic cell profiles. The method is operated by fluorescence-based detection of counterstain via laser beam-employed instruments including flow cytometer, fluorescence microscope and automated cell counter. The detection is primarily conducted based on the same principle; however the efficiency of instruments may vary. Here we evaluated the efficiency of those instruments for the clear-cut detection of cell death through various mammalian and microalgae cell lines. To the best of our knowledge, this is the first study revealing comparative analyses of apoptotic and necrotic cells in mammalian and microalgae cells using Annexin V-PI counterstain detected by flow cytometer, fluorescence microscope and automated cell counter. Fluorescence microscope and cell counter instruments were also tested and compared for the traditional trypan blue-based cell viability detection performance. For these, cell death was induced by UV-irradiation and/or bee venom for mammalian (pancreatic cancer, metastatic breast cancer and mouse fibroblasts) and microalgae cells (Chlorella vulgaris), respectfully. Findings postulated that automated cell counter and fluorescence microscopy revealed similar patterns for the detection by both counterstain and trypan blue in mammalian cells. Interestingly, flow cytometry did provide an accurate and significant detection for only one mammalian cell line when UV-treatment was followed by routine Annexin V-Propidium iodide counterstaining. Unlike, only flow cytometry revealed a significant change in the detection of death of microalgae cells by Annexin V-Propidium iodide method, but both Annexin and conventional trypan blue methods were not applicable for the automated cell counter and microscopic detections for microalgae cells. The related outputs propose that the obtaining reliable quantitation strongly depends on cell type and instruments used. These suggest the necessity of optimization and validation endeavors before any cell death detection initiative. The analytical outcomes present insights into detailed assessment of cell death detection of eukaryotic cells and provide a direction to researchers to consider.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  1. Alfraidi A, Fagundes TC (2018) Cell-based cytotoxicity methods. JAN 1(1):3–12

  2. Huang X, Chen L, Liu WJ, Qiao Q, Wu K, Wen J, Huang CH, Tang R, Zhang XZ (2015) Involvement of oxidative stress and cytoskeletal disruption in microcystin-induced apoptosis in CIK cells. Aquat Toxicol 165:41–50

    Article  CAS  Google Scholar 

  3. Xiao K, Li YP, Wang C, Ahmad S, Vu M, Kuma K, Cheng YQ, Lam KS (2015) Disulfide cross-linked micelles of novel HDAC inhibitor thailandepsin a for the treatment of breast cancer. Biomaterials 67:183–193

    Article  CAS  Google Scholar 

  4. Shan X, Tian LL, Zhang YM, Wang XQ, Yan Q, Liu JW (2015) Ginsenoside Rg3 suppresses FUT4 expression through inhibiting NF-kappa B/p65 signaling pathway to promote melanoma cell death. Int J Oncol 47(2):701–709

    Article  CAS  Google Scholar 

  5. Deng ZT, Xiao Y, Pan M, Li F, Duan WL, Meng L, Liu X, Yan F, Zheng HR (2016) Hyperthermia-triggered drug delivery from iRGD-modified temperature-sensitive liposomes enhances the anti-tumor efficacy using high intensity focused ultrasound. J Control Release 243:333–341

    Article  CAS  Google Scholar 

  6. Hwang JH, Park H, Choi DW, Nam KT, Lim KM (2018) Investigation of dermal toxicity of ionic liquids in monolayer-cultured skin cells and 3D reconstructed human skin models. Toxicol in Vitro 46:194–202

    Article  CAS  Google Scholar 

  7. Yao RS, Han DY, Sun XY, Xie Y, Wu QY, Fu CL, Yao Y, Li HJ, Li ZY, Xu KL (2018) Scriptaid inhibits cell survival, cell cycle, and promotes apoptosis in multiple myeloma via epigenetic regulation of p21. Exp Hematol 60:63–72

    Article  CAS  Google Scholar 

  8. Svystonyuk DA, Ngu JMC, Mewhort HEM, Lipon BD, Teng GT, Guzzardi DG, Malik G, Belke DD, Fedak PWM (2015) Fibroblast growth factor-2 regulates human cardiac myofibroblast-mediated extracellular matrix remodeling. J Transl Med 13:147

    Article  Google Scholar 

  9. Sevastre B, Sarpataki O, Stan RL, Taulescu M, Sevastre-Berghian AC, Olah NK, Furtuna F, Hanganu D, Hangan AC, Cenariu M, Baldea I (2017) Anticancer activity of euonymus Europaeus fruits extract on human melanoma cells. Farmacia 65(1):56–62

    Google Scholar 

  10. Turkington RC, Longley DB, Allen WL, Stevenson L, McLaughlin K, Dunne PD, Blayney JK, Salto-Tellez M, Van Schaeybroeck S, Johnston PG (2014) Fibroblast growth factor receptor 4 (FGFR4): a targetable regulator of drug resistance in colorectal cancer. Cell Death Dis 5:e1046

    Article  CAS  Google Scholar 

  11. Sahinturk V, Kacar S, Vejselova D, Kutlu HM (2018) Acrylamide exerts its cytotoxicity in NIH/3T3 fibroblast cells by apoptosis. Toxicol Ind Health 34(7):481–489

    Article  CAS  Google Scholar 

  12. Zhang G, Zhang J, Shang D, Qi B, Chen H (2015) Deoxycholic acid inhibited proliferation and induced apoptosis and necrosis by regulating the activity of transcription factors in rat pancreatic acinar cell line AR42J. In Vitro Cell Dev Biol Anim 51(8):851–856

    Article  CAS  Google Scholar 

  13. Sousa T, Castro RE, Pinto SN, Coutinho A, Lucas SD, Moreira R, Rodrigues CMP, Prieto M, Fernandes F (2015) Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties. J Lipid Res 56(11):2158–2171

    Article  CAS  Google Scholar 

  14. Li Z, Zhu S, Huang L, Shang M, Yu C, Zhu H, Han D, Huang H, Yu X, Li X (2018) Exendin-4 impairs the autophagic flux to induce apoptosis in pancreatic acinar AR42J cells by down-regulating LAMP-2. Biochem Biophys Res Commun 496(2):294–301

    Article  CAS  Google Scholar 

  15. Celik-Uzuner S, Peters L, O'Neill C (2016) Quenching of cellular autofluorescence is necessary for specific detection of DNA methylation by flow cytometry compared to microscopy-based analysis. FEBS J 283:249–250

    Google Scholar 

  16. Aubin JE (1979) Autofluorescence of viable cultured mammalian-cells. J Histochem Cytochem 27(1):36–43

    Article  CAS  Google Scholar 

  17. Billinton N, Knight AW (2001) Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Anal Biochem 291(2):175–197

    Article  CAS  Google Scholar 

  18. Andersson H, Baechi T, Hoechl M, Richter C (1998) Autofluorescence of living cells. J Microsc (Oxford) 191:1–7

    Article  CAS  Google Scholar 

  19. Nawa Y, Inami W, Miyake A, Ono A, Kawata Y, Lin S, Terakawa S (2014) Dynamic autofluorescence imaging of intracellular components inside living cells using direct electron beam excitation. Biomed Opt Express 5(2):378–386

    Article  Google Scholar 

  20. Gerken HG, Donohoe B, Knoshaug EP (2013) Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production. Planta 237(1):239–253

    Article  CAS  Google Scholar 

  21. Yap BHJ, Crawford SA, Dagastine RR, Scales PJ, Martin GJO (2016) Nitrogen deprivation of microalgae: effect on cell size, cell wall thickness, cell strength, and resistance to mechanical disruption. J Ind Microbiol Biotechnol 43(12):1671–1680

    Article  CAS  Google Scholar 

  22. Safi C, Ursu AV, Laroche C, Zebiba B, Merah O, Pontalier PY, Vaca-Garcia C (2014) Aqueous extraction of proteins from microalgae: effect of different cell disruption methods. Algal Res 3:61–65

    Article  Google Scholar 

  23. da Silva TL, Reis A, Medeiros R, Oliveira AC, Gouveia L (2009) Oil production towards biofuel from autotrophic microalgae Semicontinuous cultivations Monitorized by flow cytometry. Appl Biochem Biotechnol 159(2):568–578

    Article  Google Scholar 

  24. Montero MF, Aristizabal M, Reina GG (2011) Isolation of high-lipid content strains of the marine microalga Tetraselmis suecica for biodiesel production by flow cytometry and single-cell sorting. J Appl Phycol 23(6):1053–1057

    Article  CAS  Google Scholar 

  25. Pereira H, Schulze PSC, Schuler LM, Santos T, Barreira L, Varela J (2018) Fluorescence activated cell-sorting principles and applications in microalgal biotechnology. Algal Res 30:113–120

    Article  Google Scholar 

  26. Barteneva NS, Fasler-Kan E, Vorobjev IA (2012) Imaging flow cytometry: coping with heterogeneity in biological systems. J Histochem Cytochem 60(10):723–733

    Article  CAS  Google Scholar 

Download references

Acknowledgements

A part of this study was supported by the equipment grant (Project ID: FAY-2016-5755) of Karadeniz Technical University, Scientific Research Coordination Unit, to Dr. Uğur Uzuner. Authors would like to thank to Prof Sevgi Kolayli (Karadeniz Technical University, Department of Biochemistry) for the kindly gift of Annexin/PI detection kit, and to Prof Figen Celep Eyupoglu (Karadeniz Technical University, Department of Medical Biology) for providing a batch of MDA-MB231 cells.

Author information

Authors and Affiliations

  1. Department of Molecular Biotechnology, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey

    Emine Koç & Ramazan Çakmak

  2. Department of Molecular Biology and Genetics, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey

    Selcen Çelik-Uzuner & Uğur Uzuner

Authors
  1. Emine Koç
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Selcen Çelik-Uzuner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Uğur Uzuner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ramazan Çakmak
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

EK performed all experiments with mammalian cells, RC performed microalgae experiments. SCU designed the study and wrote the manuscript. UU edited the manuscript.

Corresponding author

Correspondence to Selcen Çelik-Uzuner.

Ethics declarations

Competing Interest

Authors declare no competing interest exists.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Koç, E., Çelik-Uzuner, S., Uzuner, U. et al. The Detailed Comparison of Cell Death Detected by Annexin V-PI Counterstain Using Fluorescence Microscope, Flow Cytometry and Automated Cell Counter in Mammalian and Microalgae Cells. J Fluoresc 28, 1393–1404 (2018). https://doi.org/10.1007/s10895-018-2306-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10895-018-2306-4

Keywords

Search

Navigation