Cell viability

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Cell viability ( cell viability , viable cell percentage , English cell viability ) referred to in the cell biology and microbiology the proportion of living cells in a cell population.

properties

The number of dead cells and the number of living cells (living cell number ) together give the total cell number. The total number of cells can e.g. B. be determined with a counting chamber under a microscope , with a flow cytometer or a Coulter counter . Cell death occurs through two mechanisms, necrosis and apoptosis . The vital staining , however, is not a method for determining the cell viability, but those processes for staining of cells without killing it.

The IC 50 value ( inhibitory concentration of half-maximal effect ) indicates the concentration of a substance which lowers cell viability by 50% in investigations into cytotoxicity . In some publications the LC 50 value ( lethal instead of inhibitory ) is also used, but in this context it is identical to the IC 50 value. This value is of interest , for example, for the effectiveness of chemotherapeutic agents or disinfectants . The smaller the necessary concentration or dose , the higher the effectiveness.

Verification procedure

There is a large number of tests on the market for determining cell viability that work according to different measurement principles. The methods include microscopic , fluorimetric , colorimetric , luminometric , flow cytometric and impedance change-based methods. In the case of colony-forming microorganisms, a surface-viable count is usually carried out .

Proof principles

Proof of viability is based on properties of living cells such as endocytosis, enzymatic activity, integrity of the cell membrane or reproduction. Since each method has weaknesses, partially fluorescent double staining are carried out for the simultaneous detection of apoptosis and necrosis. B. the vital fluorescence double staining with propidium iodide (PI) and annexin V (see below). Double-stained cells with Annexin V and PI indicate dead cells, while single PI-stained cells are classified as necrotic and single Annexin V-stained cells are classified as apoptotic. In the following, it should be noted that the evidence listed indicates either living or dead cells.

Measurement of living cells

Endocytosis evidence

This viability can be indicated , for example, in the neutral red test by the uptake of the vital dye neutral red ( neutral red uptake , NRU ) in cell lysosomes through endocytosis .

Enzymatic evidence

Microtiter plate of an MTT assay
Esterase detection with fluorescein diacetate

Furthermore, detection of enzymatic activity is carried out, which decreases in dead cells, but may still be present in some cases. These evidence therefore stain living cells.

As Esterasenachweise be CalceinAM or fluorescein diacetate used. The activity of lactate dehydrogenase (LDH) can also be measured.

The redox indicator resazurin , hydroethidine, the MTT test with 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide or its analogue XTT as well as the luciferase- based methods indirectly indicate the redox potential Glycolysis rate of living cells after.

Bromodeoxyuridine (BrdU) is used to detect DNA replication in growing cells.

The WST-1 assay ( w ater s oluble t etrazolium ) used for the detection of an intact respiratory chain into cells. Viable cells with an intact mitochondrial succinate - tetrazolium dehydrogenase system cause an enzymatic conversion of the pale red colored tetrazolium salt WST-1 (4- [3- (4-iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] - 1,3-benzene disulfonate) into the dark red formazan . This color change can be measured and evaluated photometrically in a spectrophotometer .

Proof of proliferation

The measurement of the increase in growing cells in a given period enables the viability to be determined via the growth rate and the generation time .

Total protein detection

The Kenacid blue staining of the proteins of all cells shows only a limited correlation with other methods.

Measurement of dead cells

Evidence of apoptosis

Proof of apoptosis can with annexin V , with Yo-Pro or the TUNEL assay done. Not all dead cells undergo apoptosis; in the event of mechanical overload , cells die through cracks in the cell membranes (see necrosis ).

Proof of perforation

Trypan blue staining of dead cells

Diffusion-based methods use the perforation dyes trypan blue , brilliant blue FCF , crystal violet or the DNA- intercalating fluorescent dyes 4 ′, 6-diamidine-2-phenylindole (DAPI), ethidium bromide or propidium iodide , which can penetrate through perforated cell membranes into dead cells. In contrast, living cells are hardly stained. Detection of perforations requires a rapid counting after the addition of the dye due to a (albeit significantly lower) dye diffusion, even in living, intact cells.

literature

Individual evidence

  1. ↑ Overview of measurement methods for determining cell viability and proliferation
  2. ↑ a b c d L. Schröterová, V. Králová, A. Vorácová, P. Hasková, E. Rudolf, M. Cervinka: Antiproliferative effects of selenium compounds in colon cancer cells: comparison of different cytotoxicity assays. In: Toxicol Vitro . (2009) Vol. 23 (7), pp. 1406-1411. PMID 19607906 .
  3. ^ DWR Gray, et al. : The use of fluorescein diacetate and ethidium bromide as a viability stain for isolated islets of Langerhans. In: Stain Technology 62/1987 pp. 373-381.
  4. ↑ EM Czekanska: Assessment of cell proliferation with resazurin-based fluorescent dye. In: Methods Mol Biol. (2011) Vol. 740, pp. 27-32. PMID 21468965 .
  5. ^ E. Matteucci, O. Giampietro: Flow cytometry study of leukocyte function: analytical comparison of methods and their applicability to clinical research. In: Curr Med Chem . (2008) Vol. 15 (6), pp. 596-603. Review. PMID 18336274 .
  6. ↑ a b C. Mehanna, C. Baudouin, F. Brignole-Baudouin: Spectrofluorometry assays for oxidative stress and apoptosis, with cell viability on the same microplates: a multiparametric analysis and quality control. In: Toxicol In Vitro. (2011) Vol. 25 (5), pp. 1089-1096. PMID 21419213 .
  7. ↑ VN Sumantran: Cellular chemosensitivity assays: an overview. In: Methods Mol Biol. (2011) Vol. 731, pp. 219-36. PMID 21516411 .
  8. ↑ Introduction to in-vitro testing ( Memento from June 13, 2007 in the Internet Archive )
  9. ↑ MC Didiot, S. Serafini, MJ Pfeifer, FJ King, CN Parker: Multiplexed reporter gene assays: monitoring the cell viability and the compound kinetics on luciferase activity . In: J Biomol Screen . (2011) Vol. 16 (7), pp. 786-93. PMID 21693766 .
  10. ^ Y. Lu, F. Jiang, H. Jiang, K. Wu, X. Zheng, Y. Cai, M. Katakowski, M. Chopp, SS To: Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells. In: Eur J Pharmacol . (2010) Vol. 641 (2-3), pp. 102-7. PMID 20553913 . PMC 3003697 (free full text).
  11. ^ AV Peskin, CC Winterbourn: A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). In: Clinica Chimica Acta. Vol. 293, 2000, pp. 157-166. PMID 10699430 .
  12. ↑ Zimmermann I., Collateral chemosensitivity / resistance in acute myeloid leukemia , dissertation 2006, LMU Munich
  13. ^ R. Clothier, E. Gottschalg, S. Casati, M. Balls: The FRAME alternatives laboratory database. 1. In vitro basal cytotoxicity determined by the Kenacid blue total protein assay. In: Altern Lab Anim. (2006) Vol. 34 (2), pp. 151-75. PMID 16704290 .
  14. ↑ H. Hurst, RH Clothier, M. Pratten: An evaluation of the chick cardiomyocyte micromass system for identification of teratogens in a blind trial. In: Reprod Toxicol . (2009) Vol. 28 (4), pp. 503-10. PMID 19646523 .
  15. ↑ MJ Stoddart: Cell viability assays: introduction. In: Methods Mol Biol. (2011) Vol. 740, pp. 1-6. PMID 21468961 .
  16. ↑ a b P. Chrenek, AV Makarevich, M. Simon: Viability and apoptosis in spermatozoa of transgenic rabbits. In: Zygote. (2012) Vol. 20 (1), pp. 33-7. PMID 21144118 .