Cell-based assays using calcein acetoxymethyl ester show variation in fluorescence with treatment conditions


  • Fayth Miles University of California, Los Angeles, CA
  • Jill Lynch The University of Delaware
  • Robert Sikes Laboratory for Cancer Ontogeny and Therapeutics, Department of Biological Sciences The Center for Translational Cancer Research, The University of Delaware, Newark, DE




cellular fluorescence, calcein, cell-based assays, biochemical compounds, prostate cancer cells


The use of fluorogenic compounds in cell and molecular biology has increased in both frequency and range of applications. However, such compounds may introduce artifacts in intracellular fluorescence and cell number estimations as a consequence of interaction with exogenous stimulants, necessitating the use of adequate controls for accurate measurements and valid conclusions. Using calcein acetoxymethyl ester (AM) in combination with various exogenous cellular treatments, we report that the standard practice of direct normalization of experimental values to controls is insufficient for fluorogenic measurements. Treatments applied to cells may influence intracellular conversion of the fluorogenic compound, thereby enhancing or decreasing fluorescence relative to controls. We hereby encourage caution and recommend normalization of cellular fluorescence within each treatment group before comparison to controls.

Author Biographies

Fayth Miles, University of California, Los Angeles, CA

Department of Epidemiology, Fielding School of Public Health, University of California, Los Angeles, CA

Senior Fellow Trainee

Jill Lynch, The University of Delaware

Laboratory for Cancer Ontogeny and Therapeutics, Department of Biological Sciences, The University of Delaware, Newark DE 19716

Graduate Student

Robert Sikes, Laboratory for Cancer Ontogeny and Therapeutics, Department of Biological Sciences The Center for Translational Cancer Research, The University of Delaware, Newark, DE

Department of Biological Sciences, associate professor; Center for Translational Cancer Research, director

University of Delaware, Newark, DE


Berggren S, Hoogstraate J, Fagerholm U, Lennernas H. Characterization of jejunal absorption and apical efflux of ropivacaine, lidocaine and bupivacaine in the rat using in situ and in vitro absorption models. Eur J Pharm Sci. 2004;21(4):553-60. Epub 2004/03/05.

Bratosin D, Mitrofan L, Palii C, Estaquier J, Montreuil J. Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging. Cytometry A. 2005;66(1):78-84. Epub 2005/05/26.

Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J. A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes. J Immunol Methods. 1995;178(1):41-51. Epub 1995/01/13.

Kansui Y, Garland CJ, Dora KA. Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries. Cell Calcium. 2008;44(2):135-46. Epub 2008/01/15.

Opas M, Dziak E. Intracellular pH and pCa measurement. Methods Mol Biol. 1999;122:305-13. Epub 1999/05/08.

Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, et al. Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J. 1999;76(2):725-34. Epub 1999/02/04.

So EC, Sallin MA, Zhang X, Chan SL, Sahni L, Schulze DH, et al. A high throughput method for enrichment of natural killer cells and lymphocytes and assessment of in vitro cytotoxicity. J Immunol Methods. 2013. Epub 2013/05/18.

Uggeri J, Gatti R, Belletti S, Scandroglio R, Corradini R, Rotoli BM, et al. Calcein-AM is a detector of intracellular oxidative activity. Histochem Cell Biol. 2004;122(5):499-505. Epub 2004/10/27.

Pliquett UF, Gusbeth CA. Overcoming electrically induced artifacts in penetration studies with fluorescent tracers. Bioelectrochemistry. 2000;51(1):75-9. Epub 2000/05/03.

Thalmann GN, Sikes RA, Wu TT, Degeorges A, Chang SM, Ozen M, et al. LNCaP progression model of human prostate cancer: androgen-independence and osseous metastasis. Prostate. 2000;44(2):91-103 Jul 1;44(2).

Chang SM, Chung LW. Interaction between prostatic fibroblast and epithelial cells in culture: role of androgen. Endocrinology. 1989;125(5):2719-27.

Thalmann GN, Anezinis PE, Chang SM, Zhau HE, Kim EE, Hopwood VL, et al. Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer. Cancer Res. 1994;54(10):2577-81.

Almeida-Porada G, Ascensao JL. Isolation, characterization, and biologic features of bone marrow endothelial cells. J Lab Clin Med. 1996;128(4):399-407. Epub 1996/10/01.

Sikes RA, Walls AM, Brennen WN, Anderson JD, Choudhury-Mukherjee I, Schenck HA, et al. Therapeutic approaches targeting prostate cancer progression using novel voltage-gated ion channel blockers. Clin Prostate Cancer. 2003;2(3):181-7.

Anderson JD, Hansen TP, Lenkowski PW, Walls AM, Choudhury IM, Schenck HA, et al. Voltage-gated sodium channel blockers as cytostatic inhibitors of the androgen-independent prostate cancer cell line PC-3. Mol Cancer Ther. 2003;2(11):1149-54.

Sikes RA, Nicholson BE, Koeneman KS, Edlund NM, Bissonette EA, Bradley MJ, et al. Cellular interactions in the tropism of prostate cancer to bone. Int J Cancer. 2004;110(4):497-503.

Jones TR, Kang IH, Wheeler DB, Lindquist RA, Papallo A, Sabatini DM, et al. CellProfiler Analyst: data exploration and analysis software for complex image-based screens. BMC bioinformatics. 2008;9:482. Epub 2008/11/19.

Lu S, Kim TJ, Chen CE, Ouyang M, Seong J, Liao X, et al. Computational analysis of the spatiotemporal coordination of polarized PI3K and Rac1 activities in micro-patterned live cells. PLoS One. 2011;6(6):e21293. Epub 2011/07/09.

McGowan TA, Madesh M, Zhu Y, Wang L, Russo M, Deelman L, et al. TGF-beta-induced Ca(2+) influx involves the type III IP(3) receptor and regulates actin cytoskeleton. Am J Physiol Renal Physiol. 2002;282(5):F910-20. Epub 2002/04/06.

Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, et al. LNCaP model of human prostatic carcinoma. Cancer Res. 1983;43(4):1809-18.




How to Cite

Miles F, Lynch J, Sikes R. Cell-based assays using calcein acetoxymethyl ester show variation in fluorescence with treatment conditions. J Biol Methods [Internet]. 2015Oct.29 [cited 2022Aug.11];2(3):e29. Available from: https://jbmethods.org/jbm/article/view/73