Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

Lysis of tumor cells by antibody and complement. VII. Complement-dependent 86Rb release--a nonlethal event?

The guinea pig hepatoma (line-1) treated with anti-Forssman antibody (TA) and GPC sequentially released 86Rb, 14C from 14C aminoidobutyric acid and failed to exclude trypan blue. Incubation of TA with fluid phase GPC for 1 min caused maximal 86Rb release; however, if the GPC was removed at this time, the cells were not subsequently killed. Using a number of naturally occurring human sera deficient in a complement component we have shown 86Rb release requires the binding of the complement components 1 through 8, but there was no absolute requirement for C9. Irreversible damage to the cell as measured by 14C AIB release or uptake of trypan blue required the complete sequence of complete sequence of complement components. These observations indicate that 86Rb release is not a relible indicator cytotoxicity.     

Improved sensitivity of trypan blue dye exclusion assay with Ni2+ or Co2+ salts

A modified trypan blue dye exclusion assay was developed usingNi²⁺ or Co²⁺ salts to determine the viability ofprimary and transformed cells. When the cells were preincubatedwith NiSO₄ or CoCl₂ followed by trypan blue assay, thecontrast between stained and unstained cells was significantlyincreased as compared to the conventional trypan blue dyeexclusion assay.     

Cytotoxicity as measured by trypan blue as a potentially confounding variable in the in vitro alkaline elution/rat hepatocyte assay

Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed increased level of DNA-strand breaks as determined by alkaline elution, without an appreciable increase in cytotoxicity as determined by a trypan blue dye exclusion assay at harvest. The alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often associated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analysis of hepatocytes treated with A2RA by pulsed-field gel electrophoresis and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochondria; although cell blebbing was seen using both EM and light microscopy, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan blue at harvest. Thus, whereas in our experience trypan blue dye exclusion accurately reflects cytotoxicity induced by the majority of test agents, in this rather unusual case, trypan blue did not accurately reflect compound-induced cytotoxicity at harvest since there was no concurrent loss of membrane integrity. However, when hepatocytes treated with A2RA were incubated for either 3 h or 20 h in the absence of compound, a sharp, dose-dependent decline in viability was observed using trypan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed DNA double-strand breaks arose as a consequences of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct compound-DNA interaction. Since DNA double-strand breaks behave under alkaline denaturing conditions as two single-strand breaks and can therefore produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in this assay is that cytotoxicity by trypan blue dye exclusion will not be greater than 30%. Our data, however, indicate that interpretation of the elution assay as a test for genotoxicity can still be confounded by the failure of the trypan blue dye exclusion assay to reflect cytotoxicity in the unusual instance when there is no concurrent, immediate loss of membrane integrity.     

Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus

Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.     

High radiosensitivity of the MOLT-4 leukaemic cell line

Radiation survival of MOLT-4, a leukaemic T-lymphocyte cell line, was measured by counting colonies formed in 0.8 per cent methyl cellulose. The survival curve was a simple exponential and showed the cells to be radiation sensitive, with D0 = 0.49 +/- 0.02 Gy and extrapolation number n = 0.92 +/- 0.09. No increase in survival as measured by colony-forming ability or trypan blue dye exclusion was seen when the dose was split into two fractions, separated by a 5 h incubation period. Electron microscopy and trypan blue dye exclusion showed that 5 h after exposure to high doses, MOLT-4 cells began to die and displayed condensed, marginated chromatin and cellular vesiculation.     

Estimation of Cytotoxic Injury to Gastric Parietal Cells by Trypan Blue Exclusion Test Followed by Hematoxylin-Eosin Counterstaining of Fixed Smears

A modification of a trypan blue exclusion technique for detection of cyto-toxicity is described with the use of a trypan blue exclusion test followed in sequence by hematoxylin-eosin counterstaining of smears permanently fixed. This technique, as applied to the suspension of parietal cells, permits the distinction between intact, moderately damaged, and severely damaged cells, determination of their ratio to each other, and an insight into the interrelations of aggressive cells (sensitized lymphocytes) to target cells (parietal cells) in the permanently fixed gastric cells suspension.     

Label-free measurement of cell viability via counting cells attached on affinity substrates

The commonly used trypan blue dye exclusion method and other modified cell viability methods, such as fluorescein dye and tetrazolium dye exclusion, artificially introduce toxic chemicals to cells and, thus, alter cellular organelles when measuring cell viability. Therefore, cell viability could be affected by the processes currently used to observe viability. In this study, the cell viability of Chinese hamster ovary (CHO) cells was measured by simply counting attached cells after the cultured CHO cells were attached on a Concanavalin A (Con A) substrate. The efficiency of cell attachment to Con A surfaces was different for live and dead cells allowing the cell viability of CHO cells to be measured without any chemical modifications to the cells.     

MTT法用于药物对L2细胞毒性的实验研究

目的：评价噻唑蓝（MTT）法检测药物对细胞的毒性作用的可靠性。方法：大鼠肺泡上皮L2细胞以叔丁基对苯二酚（TBHQ）10．100μM,BsO以1-10mM分别处理,用MTT法检测细胞活性、JC-1（5,5’,6,6’-四氯．1,1’,3,3’-四乙基苯并咪唑羰花青碘化物）荧光染料法检测细胞线粒体电位改变、台盼蓝排斥实验检测细胞死亡率,分析各指标的情况。结果：在处理剂量范围,MTT法检测到的光密度（OD）值未能达到一般判断的半数抑制浓度（ic50）水平,最高抑制率仅达到30％左右;台盼蓝排斥试验检测数据表明TBHQ的LC50值为50μM,丁硫氨酸亚砜胺（BSO）为5mM;利用JC-1荧光染料判断的半数凋亡剂量分别为50μM和7mM。结论：MTT法作为最常采用的细胞生长抑制检测手段,但在某些特定实验中可能不能客观地反映细胞的活性,建议多种方法结合进行评价。     

Isolation of human hepatocytes after hepatic warm and cold ischemia: a practical approach using University of Wisconsin solution

A method is described for isolating human hepatocytes from tissue fragments after warm and cold ischemia as experienced during hepatic resections. Cells with a high trypan blue dye exclusion and good culture characteristics were isolated by employing an initial tissue perfusion with UW solution. The method could facilitate transfer of liver tissues between distant centers for cell isolation studies.     

A simple and rapid method for evaluating the survival of xeroderma pigmentosum lymphoid lines after irradiation with ultraviolet light

A simple, rapid, and reproducible test has been developed to measure the viability of cells after irradiation with ultraviolet light (UV). Epstein-Barr virus-transformed lymphoid lines, derived from patients with xeroderma pigmentosum (XP), were irradiated with UV, and the post-UV viability of the lymphoid lines was determined by the trypan blue dye exclusion method. The relative post-UV survival of the patients' lymphoid lines was similar to the relative post-UV survival of the patients' fibroblast strains.     

A new methodology for plant cell viability assessment using intracellular esterase activity

 A plant cell suspension culture of Alfalfa (Medicago sativa L.) was grown in a bioreactor using a batch procedure. The cytoplasmic esterase activity (EC 3.1) was extracted from the cells and measured during cultivation using fluorescein diacetate as the fluorogenic substrate. This enzymatic activity was conclusively found to be correlated to cell viability assessed with the membrane integrity test using the trypan blue dye. This new viability determination method is convenient, simple and can be reproduced because: (1) the difficult step of counting the cells when using the trypan blue exclusion method is avoided and (2) the esterase activity level per viable cell constituted of numerous enzymes depends on cell viability but is independent of cellular metabolism. Received: 28 January 1999 / Accepted: 1 April 1999     

A simple and rapid method for evaluating the survival of xeroderma pigmentosum lymphoid lines after irradiation with ultraviolet light

Summary A simple, rapid and reproducible test has been developed to measure the viability of cells after irradiation with ultraviolet light (UV). Epstein-Barr virus-transformed lymphoid lines, derived from patients with xeroderma pigmentosum (XP), were irradiated with UV, and the post-UV viability of the lymphoid lines was determined by the trypan blue dye exclusion method. The relative post-UV survival of the patients' lymphoid lines was similar to the relative post-UV survival of the patients' fibroblast strains.     

Demonstration of fluorescent X and Y bodies after antibody- and complement-mediated cytotoxicity

A technique for fluorescence staining of X and Y bodies (sex chromatin) after antibody- and complement-mediated cytotoxicity test has been developed. Cytotoxicity was quantitated by staining the dead cells with trypan blue (dye exclusion test). X bodies (Barr bodies) of human female fibroblast (stained with acridine orange) were observed in about 40 percent of the cells which survived cytotoxicity. Y bodies were studied on human male fibroblasts and in a hamster/human hybrid line which retained the human Y chromosome only. Fluorescent Y body was detectable in from 50 to 60 percent of the cells which survived the serological test. The double staining procedure did not significantly affect the proportion of killed (trypan blue-positive) cells. We suggest that this is a useful method for the detection of cytotoxic antibodies against the products of X and Y chromosomes, especially when mixed cell populations-such as tumor, sex chromosome mosaics, sperm, and artificially mixed human male and female cell lines-are tested.     

Cytotoxic effects of FK506 on human renal proximal tubule cells in culture

Summary FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200μg/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40μg/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.     

Effect of extracellularly generated free radicals on the plasma membrane permeability of isolated pancreatic B-cells

Previous experiments on alloxan diabetogenicity suggest that alloxan increases the permeability of B-cell plasma membranes by generation of noxious free radicals. Whether the radicals are generated intra- or extracellularly has however been disputed. To test if extracellularly generated free radicals could decrease trypan blue exclusion of dispersed islet cells, a radical-generating solution of xanthine oxidase/hypoxanthine was employed. The solution increased dye uptake by cells in the cell suspension. Superoxide dismutase and catalase but not scavengers of hydroxyl radicals protected against the increase in dye uptake. Both L- and D-glucose protected the cells from injury. It is concluded that extracellularly generated free radicals induce damage to the plasma membrane of islet cells. The result strengthens the hypothesis of plasma membrane damage by extracellularly generated free radicals as the primary event in alloxan diabetogenicity and may provide a link for explanation of damage caused by islet inflammation in juvenile diabetes.     

Evidence for inactivation of DNA repair in frozen and thawed mammalian cells.

A variety of cell strains and lines were frozen and thawed by conventional techniques for cell storage. Following thawing, extracts of cells were prepared and incubated with UV-irradiated E. coli DNA. Thymine dimer excision activity present in extracts of unfrozen cells was lost in extracts of recently thawed cells. The ability to exercise dimers was restored after about 40 h post-thawing, but the recovery was inhibited if cells were cultured in the presence of puromycin. Correlating with the loss of dimer excising activity there was a reduced cell viability as measured by trypan blue dye exclusion.     

The mechanism of action of low doses of gamma radiation connected with the development of human cell resistance to mutagens]

The human fibroblasts were gamma-irradiated with low doses (0.07-0.21 Gy). After a short time interval (3 h), a study was made of the postirradiation viability of cells (by the trypan blue dye exclusion method); post-N-methyl-N'-nitro-nitrosoguanidine-DNA synthesis (by 3H-thymidine incorporation immediately after the mutagen treatment) and postirradiation induction of DNA single-strand breaks (by alkaline elution of cells lysed on the membrane filters). The preirradiation of cells with low doses of gamma-rays was shown to render the cells resistance to induction of DNA breaks by the following exposure to gamma-radiation. The survival rate increased; DNA synthesis was resistant to alkylation damage in these cells, as compared to nonirradiated cells.     

In vitro evaluation of a toxic metabolite of sulfadiazine

We have demonstrated the in vitro production of a potentially toxic metabolite of sulfadiazine Human lymphocytes were incubated with sulfadiazine and a murine hepatic microsomal drug metabolizing system. Toxicity to cells was assessed by trypan blue dye exclusion. Covalent binding of labelled sulfadiazine to microsomes also was studied. Sulfadiazine toxicity to cells was dependent on microsomes and NADPH. Binding and toxicity were decreased when microsomes were boiled or cytochrome P-450 inhibited, and by the addition of N-acetylcysteine or glutathione. The data suggest the production of a toxic intermediate of oxidative metabolism of sulfadiazine which is detoxified by conjugation with glutathione. Covalent binding of such metabolites to cell macromolecules could lead to cell death and, by acting as haptens, to secondary hypersensitivity reactions.     

Dynamics of vitellogenin uptake in Aedes aegypti as demonstrated by trypan blue

Trypan blue has been shown to be a reliable indicator of the micropinocytotic uptake of vitellogenin by developing oöcytes. Trypan blue was injected into the mosquito Aedes aegypti to determine at what times after the blood meal vitellogenin was taken up. Histological sections examined by light microscopy showed that trypan blue began to be sequestered from 2 to 5 hr after the blood meal. Any association between dye and ovariole ended from 39 to 42 hr after the blood meal, in which period no dye was incorporated into spheres of yolk protein. Of the times investigated in this experiment, the greatest amount of dye was seen in the oöcyte at 24 hr after the blood meal. The onset and conclusion of trypan blue uptake correspond with the related events in the synthesis of vitellogenin by the fat body. The presence of trypan blue in occasional interfollicular spaces suggests that the route of entry of vitellogenin in Aedes aegypti is indeed an interfollicular one.