Abstract
A decrease in flow cytometric forward light scatter (FSC) is commonly interpreted as a sign of apoptotic cell volume decrease (AVD). However, the intensity of light scattering depends not only on the cell size but also on its other characteristics, such as hydration, which may affect the scattering in the opposite way. That makes estimation of AVD by FSC problematic. Here, we aimed to clarify the relationship between light scattering, cell hydration (assayed by buoyant density) and cell size by the Coulter technique. We used human lymphoid cells U937 exposed to staurosporine, etoposide or hypertonic stress as an apoptotic model. An initial increase in FSC was found to occur in apoptotic cells treated with staurosporine and hypertonic solutions; it is accompanied by cell dehydration and is absent in apoptosis caused by etoposide that is consistent with the lack of dehydration in this case. Thus, the effect of dehydration on the scattering signal outweighs the effect of reduction in cell size. The subsequent FSC decrease, which occurred in parallel to accumulation of annexin-positive cells, was similar in apoptosis caused by all three types of inducers. We conclude that an increase, but not a decrease in light scattering, indicates the initial cell volume decrease associated with apoptotic cell dehydration.
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References
Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y (2000) Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci U S A 97:9487–9492. doi:10.1073/pnas.140216197
Bortner CD, Cidlowski JA (2011) Life and death of lymphocytes: a volume regulation affair. Cell Physiol Biochem 28:1079–1088. doi:10.1159/000335864
Bortner CD, Scoltock AB, Sifre MI, Cidlowski JA (2012) Osmotic stress resistance imparts acquired anti-apoptotic mechanisms in lymphocytes. J Biol Chem 287:6284–6295. doi:10.1074/jbc.M111.293001
Bortner CD, Cidlowski JA (2014) Ion channels and apoptosis in cancer. Philos Trans R Soc B 369:20130104. doi: 10.1098/rstb.2013.0104
Dezaki K, Maeno E, Sato K, Akita T, Okada Y (2012) Early-phase occurrence of K+ and Cl– efflux in addition to Ca2+ mobilization is a prerequisite to apoptosis in HeLa cells. Apoptosis 17:821–831. doi:10.1007/s10495-012-0716-3
Lang F, Hoffmann EK (2012) Role of ion transport in control of apoptotic cell death. Compr Physiol 2:2037–2061. doi:10.1002/cphy.c110046
Wlodkowic D, Telford W, Skommer J, Darzynkiewic Z (2011) Apoptosis and beyond: cytometry in studies of programmed cell death. Methods Cell Biol 103:55–98. doi:10.1016/B978-0-12-385493-3.00004-8
Wlodkowic D, Skommer J, Darzynkiewic Z (2012) Cytometry of apoptosis. Historical perspective and new advances. Exp Oncol 34:255–262
Vanden-Berghe T, Grootjans S, Goossens V, Dondelinger Y, Krysko DV, Takahashi N, Vandenabeele P (2013) Determination of apoptotic and necrotic cell death in vitro and in vivo. Methods 61:117–129. doi:10.1016/j.ymeth.2013.02.011
Vorobjev IA, Barteneva NS (2015) Temporal heterogeneity metrics in apoptosis induced by anticancer drugs. J Histochem Cytochem 63:494–510. doi:10.1369/0022155415583534
Vorobjev IA, Barteneva NS (2017) Multi-parametric imaging of cell heterogeneity in apoptosis analysis. Methods 112:105–123. doi:10.1016/j.ymeth.2016.07.003
Banfalvi G (2017) Methods to detect apoptotic cell death. Apoptosis 22:306–323. doi:10.1007/s10495-016-1333-3
Shapiro HM (2003) Practical flow cytometry. 4th edn. Wiley-Liss, New York
Yurinskaya V, Goryachaya T, Guzhova I, Moshkov A, Rozanov Y, Sakuta G, Shirokova A, Shumilina E, Vassilieva I, Lang F, Vereninov A (2005) Potassium and sodium balance in U937 cells during apoptosis with and without cell shrinkage. Cell Physiol Biochem 16:155–162. doi:10.1159/000089841
Yurinskaya VE, Moshkov AV, Rozanov YM, Shirokova AV, Vassilieva IO, Shumilina EV, Lang F, Volgareva EV, Vereninov AA (2005) Thymocyte K+, Na+ and water balance during dexamethasone- and etoposide-induced apoptosis. Cell Physiol Biochem 16:15–22. doi:10.1159/000087727
Yurinskaya VE, Rubashkin AA, Shirokova AV, Vereninov AA (2011) Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells in hypertonic medium. Cell Tissue Biol 5:487–494. doi:10.1134/S1990519X11050129
Vereninov AA, Rubashkin AA, Goryachaya TS, Moshkov AV, Rozanov YM, Shirokova AV, Strelkova EG, Lang F, Yurinskaya VE (2008) Pump and channel K (Rb+) fluxes in apoptosis of human lymphoid cell line U937. Cell Physiol Biochem 22:187–194. doi:10.1159/000149796
Vereninov AA, Volgareva EV, Matveev VV, Moshkov AV, Rozanov I, Shirokova AV, Yurinskaya VE (2003) Water and ion balance in rat thymocytes under apoptosis induced with dexamethasone or etoposide. Iono-osmotic model of cell volume decrease. Tsitologiia 45:500–509 (in Russian)
Vereninov AA, Goryachaya TS, Matveev VV, Moshkov AV, Rozanov YuM, Sakuta GA, Shirokova AV, Yurinskaya VE (2004) Cell shrinkage during apoptosis is not obligatory. Apoptosis of U937 cells induced by staurosporine and etoposide. Tsitologiia 46:609–619 (in Russian)
Yurinskaya VE, Moshkov AV, Wibberley AV, Lang F, Model MA, Vereninov AA (2012) Dual response of human leukemia U937 cells to hypertonic shrinkage: initial regulatory volume increase (RVI) and delayed apoptotic volume decrease (AVD). Cell Physiol Biochem 30:964–973. doi:10.1159/000341473
Yurinskaya VE, Rubashkin AA, Vereninov AA (2011) Balance of unidirectional monovalent ion fluxes in cells undergoing apoptosis: why does Na+/K+ pump suppression not cause cell swelling? J Physiol 589:2197–2211. doi:10.1113/jphysiol.2011.207571
Barteneva NS, Fasler-Kan E, Bernimoulin M, Stern JNH, Ponomarev ED, Duckett L, Vorobjev IA (2013) Circulating microparticles: square the circle. BMC Cell Biol 14:23. doi:10.1186/1471-2121-14-23
Atkin-Smith GK, Tixeira R, Paone S, Mathivanan S, Collins C, Liem M, Goodall KJ, Ravichandran KS, Hulett MD, Poon IKh (2015) A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure. Nat Commun 6:7439. doi:10.1038/ncomms8439
McGann LE, Walterson ML, Hogg LM (1988) Light scattering and cell volumes in osmotically stressed and frozen-thawed cell. Cytometry 9:33–38. doi:10.1002/cyto.990090106
Sloot PMA, Hoekstra AG, Figdor CG (1988) Osmotic response of lymphocytes measured by means of forward light scattering: theoretical considerations. Cytometry 9:636–641. doi:10.1002/cyto.990090620
Kerr JF (1971) Shrinkage necrosis: a distinct mode of cellular death. J Pathol 105:13–20. doi:10.1002/path.1711050103
Bortner CD, Cidlowski JA (1996) Absence of volume regulatory mechanisms contributes to the rapid activation of apoptosis in thymocytes. Am J Physiol 271:C950–C961
Maeno E, Takahashi N, Okada Y (2006) Dysfunction of regulatory volume increase is a key component of apoptosis. FEBS Lett 580:6513–6517. doi:10.1016/j.febslet.2006.10.074
Numata T, Sato K, Okada Y, Wehner F (2008) Hypertonicity-induced cation channels rescue cells from staurosporine-elicited apoptosis. Apoptosis 13:895–903. doi:10.1007/s10495-008-0220-y
Lang F, Hoffmann EK (2013) CrossTalk proposal: cell volume changes are an essential step in the cell death machinery. J Physiol 591:6119–6121. doi:10.1113/jphysiol.2013.258632
Orlov SN, Model MA, Grygorczyk R (2013) CrossTalk opposing view: the triggering and progression of the cell death machinery can occur without cell volume perturbations. J Physiol 591:6123–6125. doi:10.1113/jphysiol.2013.258624
Kasim NR, Kuželová K, Holoubek A, Model MA (2013) Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease. Apoptosis 18:521–532. doi:10.1007/s10495-013-0804-z
Model MA, Schonbrun E (2013) Optical determination of intracellular water in apoptotic cells. J Physiol 591:5843–5849. doi:10.1113/jphysiol.2013.263228
Model MA (2014) Possible causes of apoptotic volume decrease: an attempt at quantitative review. Am J Physiol Cell Physiol 306:C417–C424. doi:10.1152/ajpcell.00328.2013
Tzur A, Moore JK, Jorgensen P, Shapiro HM, Kirschner MW (2011) Optimizing optical flow cytometry for cell volume-based sorting and analysis. PLoS ONE 6(1):e16053. doi:10.1371/journal.pone.0016053
Vecsler M, Lazar I, Tzur A (2013) Using standard optical flow cytometry for synchronizing proliferating cells in the G1 phase. PLoS ONE 8:e83935. doi:10.1371/journal.pone.0083935
Ormerod MG, Paul F, Cheetham M, Sun X-M (1995) Discrimination of apoptotic thymocytes by forward light scatter. Cytometry 21:300–304. doi:10.1002/cyto.990210311
Bortner CD, Sifre MI, Cidlowski JA (2008) Cationic gradient reversal and cytoskeleton-independent volume regulatory pathways define an early stage of apoptosis. J Biol Chem 283:7219–7229. doi:10.1074/jbc.M707809200
Franco R, DeHaven WI, Sifre MI, Bortner CD, Cidlowski JA (2008) Glutathione depletion and disruption of intracellular ionic homeostasis regulate lymphoid cell apoptosis. J Biol Chem 283:36071–36087. doi:10.1074/jbc.M807061200
Mulvey CS, Zhang K, Liu WB, Waxman DJ, Bigio IJ (2011) Wavelength-dependent backscattering measurements for quantitative monitoring of apoptosis, Part 2: early spectral changes during apoptosis are linked to apoptotic volume decrease. J Biomed Opt 16:117002. doi:10.1117/1.3644911
Acknowledgements
This study was supported by Russian Foundation for Basic Research, Projects No. 15-04-00776a and No. 17-00-00364 KOMFI.
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Yurinskaya, V., Aksenov, N., Moshkov, A. et al. A comparative study of U937 cell size changes during apoptosis initiation by flow cytometry, light scattering, water assay and electronic sizing. Apoptosis 22, 1287–1295 (2017). https://doi.org/10.1007/s10495-017-1406-y
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DOI: https://doi.org/10.1007/s10495-017-1406-y