Skip to main content

Advertisement

SpringerLink
Log in

Different Cell Viability Assays Reveal Inconsistent Results After Bleomycin Electrotransfer In Vitro

  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

The aim of this study was to compare different and commonly used cell viability assays after CHO cells treatment with anticancer drug bleomycin (20 nM), high voltage (HV) electric pulses (4 pulses, 1200 V/cm, 100 µs, 1 Hz), and combination of bleomycin and HV electric pulses. Cell viability was measured using clonogenic assay, propidium iodide (PI) assay, MTT assay, and employing flow cytometry modality to precisely count cells in definite volume of the sample (flow cytometry assay). Results showed that although clonogenic cell viability drastically decreased correspondingly to 57 and 3 % after cell treatment either with HV pulses or combination of bleomycin and HV pulses (bleomycin electrotransfer), PI assay performed ~15 min after the treatments indicated nearly 100 % cell viability. MTT assay performed at 6–72 h time points after these treatments revealed that MTT cell viability is highly dependent on evaluation time point and decreased with later evaluation time points. Nevertheless, in comparison to clonogenic cell viability, MTT cell viability after bleomycin electrotransfer at all testing time points was significantly higher. Flow cytometry assay if used at later times, 2–3 days after the treatment, allowed reliable evaluation of cell viability. In overall, our results showed that in order to estimate cell viability after cell treatment with combination of the bleomycin and electroporation the most reliable method is clonogenic assay. Improper use of PI and MTT assays can lead to misinterpretation of the experimental results.

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

Similar content being viewed by others

References

  • Angius F, Floris A (2015) Liposomes and MTT cell viability assay: an incompatible affair. Toxicol In Vitro 29(2):314–319

    Article  CAS  PubMed  Google Scholar 

  • Belehradek M, Domenge C, Luboinski B, Orlowski S, Belehradek Jr J, Mir LM (1993) Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial. Cancer 72(12):3694–3700

    Article  CAS  PubMed  Google Scholar 

  • Bennett WF, Tieleman DP (2014) The importance of membrane defects-lessons from simulations. Acc Chem Res 47(8):2244–2251

    Article  CAS  PubMed  Google Scholar 

  • Berridge MV, Tan AS (1993) Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys 303:474–482

    Article  CAS  PubMed  Google Scholar 

  • Berridge MV, Tan AS, McCoy KD, Wang R (1996) The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica 4:14–19

    Google Scholar 

  • Böckmann RA, Groot BL, Kakorin S, Neumann E, Grubmüller H (2008) Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations. Biophys J 95:1837–1850

    Article  PubMed Central  PubMed  Google Scholar 

  • Casciola M, Bonhenry D, Liberti M, Apollonio F, Tarek M (2014) A molecular dynamic study of cholesterol rich lipid membranes: comparison of electroporation protocols. Bioelectrochemistry 100:11–17

    Article  CAS  PubMed  Google Scholar 

  • Cepurniene K, Ruzgys P, Treinys R, Satkauskiene I, Satkauskas S (2010) Influence of plasmid concentration on DNA electrotransfer in vitro using high-voltage and low-voltage pulses. J Membr Biol 236(1):81–85

    Article  CAS  PubMed  Google Scholar 

  • Corovic S, Al Sakere B, Haddad V, Miklavcic D, Mir LM (2008) Importance of contact surface between electrodes and treated tissue in electrochemotherapy. Technol Cancer Res Treat 7(5):393–400

    Article  PubMed  Google Scholar 

  • Edhemovic I, Brecelj E, Gasljevic G et al (2014) Intraoperative electrochemotherapy of colorectal liver metastases. Surg Oncol 110:320–327

    Article  Google Scholar 

  • Frandsen SK, Gissel H, Hojman P, Tramm T, Eriksen J, Gehl J (2012) Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis. Cancer Res 72(6):1336–1341

    Article  CAS  PubMed  Google Scholar 

  • Frandsen SK, Gissel H, Hojman P, Eriksen J, Gehl J (2014) Calcium electroporation in three cell lines: a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions. Biochim Biophys Acta 1840(3):1204–1208

    Article  CAS  PubMed  Google Scholar 

  • Gabriel B, Teissié J (1995) Spatial compartmentation and time resolution of photooxidation of a cell membrane probe in electropermeabilized Chinese hamster ovary cells. Eur J Biochem 228(3):710–718

    Article  CAS  PubMed  Google Scholar 

  • Gehl J, Skovsgaard T, Mir LM (1998) Enhancement of cytotoxicity by electropermeabilization: an improved method for screening drugs. Anticancer Drugs 9(4):319–325

    Article  CAS  PubMed  Google Scholar 

  • Gumulec J, Fojtu M, Raudenska M et al (2014) Modulation of induced cytotoxicity of doxorubicin by using apoferritin and liposomal cages. Int J Mol Sci 15(12):22960–22977

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hecht SM (2000) Bleomycin: new perspectives on the mechanism of action. J Nat Prod 63(1):158–168

    Article  CAS  PubMed  Google Scholar 

  • Hibino M, Itoh H, Kinosita K Jr (1993) Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential. Biophys J 64:1789–1800

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kaminska I, Kotulska M, Stecka A et al (2012) Electroporation-induced changes in normal immature rat myoblasts (H9C2). Gen Physiol Biophys 31(1):19–25

    Article  CAS  PubMed  Google Scholar 

  • Kinosita K Jr, Tsong TY (1978) Voltage-induced changes in the conductivity of erythrocyte membranes. Biophys J 24:373–375

    Article  PubMed Central  PubMed  Google Scholar 

  • Kumar M, Kaur P, Kumar S, Kaur S (2015) Antiproliferative and apoptosis inducing effects of non-polar fractions from Lawsonia inermis L. in cervical (HeLa) cancer cells. Physiol Mol Biol Plants 21(2):249–260

    Article  CAS  PubMed  Google Scholar 

  • Labanauskiene J, Satkauskas S, Kirveliene V, Venslauskas M, Atkocius V, Didziapetriene J (2009) Enhancement of photodynamic tumor therapy effectiveness by electroporation in vitro. Medicina 45:372–377

    PubMed  Google Scholar 

  • Li L, Tan H, Gu Z, Liu Z, Geng Y, Liu Y, Tong H, Tang Y, Qiu J, Su L (2014) Heat stress induces apoptosis through a Ca2+-mediated mitochondrial apoptotic pathway in human umbilical vein endothelial cells. Plos One 9(12):e111083

    Article  PubMed Central  PubMed  Google Scholar 

  • Miklavcic D, Beravs K, Semrov D, Cemazar M, Demsar F, Sersa G (1998) The importance of electric field distribution for effective in vivo electroporation of tissues. Biophys J 74(5):2152–2158

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Miklavčič D, Mali B, Kos B, Heller R, Serša G (2014) Electrochemotherapy: from the drawing board into medical practice. Biomed Eng Online 13(1):29. doi:10.1186/1475-925X-13-29

    Article  PubMed Central  PubMed  Google Scholar 

  • Mir LM, Banoun H, Paoletti C (1988) Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol. Exp Cell Res 175(1):15–25

    Article  CAS  PubMed  Google Scholar 

  • Mir LM, Orlowski S, Belehradek J Jr, Paoletti C (1991) Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses. Eur J Cancer 27(1):68–72

    Article  CAS  PubMed  Google Scholar 

  • Mir LM, Tounekti O, Orlowski S (1996) Bleomycin: revival of an old drug. Gen Pharmacol 27(5):745–748

    Article  CAS  PubMed  Google Scholar 

  • Mir LM, Gehl J, Sersa G et al (2006) Standard operating procedures of the electrochemotherapy. Eur J Cancer Suppl 4:14–25

    Article  Google Scholar 

  • Miyaki M, Morohashi S, Ono T (1973) Single strand scission and repair of DNA in bleomycin-sensitive and resistant rat ascites Hepatoma cells. Antibiot (Tokyo) 26(7):369–373

    Article  CAS  Google Scholar 

  • Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1:841–845

    PubMed Central  CAS  PubMed  Google Scholar 

  • Park IK, Kang DH (2013) Effect of Electropermeabilization by ohmic heating for inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes in buffered peptone sater and apple juice. Appl Environ Microbiol 79(23):7122–7129

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Poddevin B, Orlowski S, Belehradek J Jr, Mir LM (1991) Very high cytotoxicity of bleomycin introduced into the cytosol of cells in culture. Biochem Pharmacol 42(Suppl):S67–S75

    Article  CAS  PubMed  Google Scholar 

  • Pucihar G, Kotnik T, Miklavcic D, Teissié J (2008) Kinetics of transmembrane transport of small molecules into electropermeabilized cells. Biophys J 95:2837–2848

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rols MP, Teissié J (1990) Electropermeabilization of mammalian cells Quantitative analysis of the phenomenon. Biophys J 58(5):1089–1098

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Romeo S, Wu YH, Levine ZA, Gundersen MA, Vernier TP (2013) Water influx and cell swelling after nanosecond electropermeabilization. Biochim Biophys Acta 1828:1715–1722

    Article  CAS  PubMed  Google Scholar 

  • Saczko J, Kamińska I, Kotulska M, Bar J, Choromańska A, Rembiałkowska N, Bieżuńska-KK, Rossowska J, Nowakowska D, Kulbacka J (2014) Combination of therapy with 5-fluorouracil and cisplatin with electroporation in human ovarian carcinoma model in vitro. Biomed Pharmacother 68(5):573–580

    Article  CAS  PubMed  Google Scholar 

  • Satkauskas S, Andre F, Bureau MF, Scherman D, Miklavcic D, Mir LM (2005) Electrophoretic component of electric pulses determines the efficacy of in vivo DNA electrotransfer. Hum Gene Ther 16:1194–1201

    Article  CAS  PubMed  Google Scholar 

  • Sersa G, Cemazar M, Miklavcic D (1995) Antitumor effectiveness of electrochemotherapy with cis-diamminedichloroplatinum(II) in mice. Cancer Res 55(15):3450–3455

    CAS  PubMed  Google Scholar 

  • Smith KC, Weaver JC (2011) Transmembrane molecular transport during versus after extremely large, nanosecond electric pulses. Biochem Biophys Res Commun 412(1):8–12

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Soden DM, Larkin JO, Collins CG et al (2006) Successful application of targeted electrochemotherapy using novel flexible electrodes and low dose bleomycin to solid tumours. Cancer Lett 232(2):300–310

    Article  CAS  PubMed  Google Scholar 

  • Teissie J (2014) Electropermeabilization of the cell membrane. Methods Mol Biol 1121:25–46

    Article  CAS  PubMed  Google Scholar 

  • Valic B, Golzio M, Pavlin M et al (2003) Effect of electric field induced transmembrane potential on spheroidal cells: theory and experiment. Eur Biophys 32(6):519–528

    Article  Google Scholar 

  • Vásquez JL, Gehl J, Hermann GG (2012) Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line: a potential improvement of intravesical chemotherapy in bladder cancer. Bioelectrochemistry 88:127–133

    Article  PubMed  Google Scholar 

  • Venslauskas MS, Satkauskas S, Rodaite-Riseviciene R (2009) Efficiency of the delivery of small charged molecules into cells in vitro. Bioelectrochemistry 79(1):130–135

    Article  PubMed  Google Scholar 

  • Weaver JC, Chizmadzhev YA (1996) Theory of electroporation: a review. Bioelectrochem Bioenerg 41:135–160

    Article  CAS  Google Scholar 

  • Yarmush ML, Golberg A, Serša G, Kotnik T, Miklavčič D (2014) Electroporation-based technologies for medicine: principles, applications, and challenges. Annu Rev Biomed Eng 16:295–320

    Article  CAS  PubMed  Google Scholar 

  • Zou Y, Wang C, Peng R, Wang L, Hu X (2015) Theoretical analyses of cellular transmembrane voltage in suspensions induced by high-frequency fields. Bioelectrochemistry 102:64–72

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was funded by Research Council of Lithuania Project TAP-03/2012 (scientific cooperation of Lithuania, Latvia, and Taiwan).

Author information

Authors and Affiliations

  1. Biophysical Research Group, Faculty of Natural Sciences, Vytautas Magnus University, Vileikos 8, 44404, Kaunas, Lithuania

    Baltramiejus Jakštys, Paulius Ruzgys, Mindaugas Tamošiūnas & Saulius Šatkauskas

Authors
  1. Baltramiejus Jakštys
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paulius Ruzgys
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mindaugas Tamošiūnas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Saulius Šatkauskas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Saulius Šatkauskas.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jakštys, B., Ruzgys, P., Tamošiūnas, M. et al. Different Cell Viability Assays Reveal Inconsistent Results After Bleomycin Electrotransfer In Vitro. J Membrane Biol 248, 857–863 (2015). https://doi.org/10.1007/s00232-015-9813-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00232-015-9813-x

Keywords

Search

Navigation