Evaluation of methods for determining 6-hydroxydopamine cytotoxicity

Summary The toxic effects of 6-hydroxydopamine on the human neuroblastoma cell line SK-N-SH-SY5Y (SY5Y) and the Chinese hamster ovary (CHO) cell line were measured with five viability assays. Four of the assays (attachment efficiency, plating efficiency, amino acid incorporation into acid-precipitable proteins, and Trypan Blue dye exclusion) showed higher drug susceptibility in SY5Y cells than CHO cells. Only growth inhibition (proliferation index) gave results indicating greater sensitivity in CHO cells. Over a time span of 48 hr, injured cell populations lost vital functions in the following order: attachment ability, amino acid incorporation, proliferative capacity, and dye exclusion. Recovery of each of the functions occurred in sublethally injured populations. Monitoring the extinction and recovery of vital functions permitted the accurate determination of a drug concentration (30 μg/ml) selectively toxic for SY5Y cells. A strong correlation was noted between relative values for amino acid incorporation 3 hr after drug treatment, attachment efficiency at 24 hr, and dye exclusion at 24 and 48 hr. We concluded that Trypan Blue dye exclusion and amino acid incorporation were suitable methods for comparing the effects of cytotoxins on different cell lines, provided they were performed at the appropriate time after treatment with the toxin. The combined techniques yield both population and individual cell data, are simple to do, and are applicable to nondividing cell populations. This work was supported by an NIH National Research Service Award GM07204 to E. T. C., a gift from the Lola-Wright Foundation, NINCDS Grants NS14034 and NS15234, Robert Welch Grant H698, and an RCDA (NS00213) to J. R. P.     

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.     

Dairy bacteria remove in vitro dietary lectins with toxic effects on colonic cells

Aims:  To assess in vitro the ability of some dairy bacteria to bind concanavalin A (Con A), peanut agglutinin (PNA) and jacalin (AIL), preventing their toxicity on mouse intestinal epithelial cells (IEC).
Methods and Results:  Con A and AIL reduced significantly IEC viability in vitro , as determined by Trypan Blue dye exclusion or by propidium iodide/fluorescein diacetate/Hoescht staining. Different strains of dairy bacteria were able to remove lectins from the media. Two strains were subjected to treatments used to remove S-layer, cell wall proteins, polysaccharides and lectin-like adhesins. They were then assayed for the ability to bind dietary lectins and reduce toxicity against IEC and to adhere to IEC after interaction with lectins. Con A and AIL were removed by Propionibacterium acidipropionici and Propionibacterium freudenreichii by binding with specific sugar moieties on the bacterial surface. Removal of lectins by bacteria impaired IEC protection. Adhesion of P. acidipropionici to IEC was reduced but not abolished after binding Con A or AIL.
Conclusions:  Removal of Con A or AIL by dairy propionibacteria was effective to avoid the toxic effect against colonic cells in vitro.
Significance and Impact of the Study:  Consumption of foods containing these bacteria would be a tool to protect the intestinal epithelia.     

A rapid cell membrane permeability test using flourescent dyes and flow cytometry

A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein—fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.Abbreviations DMA dimethyl sulfate - DMSO dimethyl sulfoxide - EB ethidium bromide - F fluorescein - FDA fluorescein diacetate - FS₂₅ concentration of test substance resulting in a F-peak left-shift of 25% from control - PBS phosphate buffered saline - SCT forward light scatter - SDS sodium dodecyl sulfate     

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     

Filtroporation: A simple, reliable technique for transfection and macromolecular loading of cells in suspension.

Cultured Chinese hamster ovary (CHO) cells suspended in their growth medium were forced by gas pressure through the uniformly sized micropores of filter membranes. This procedure caused transient damage to the plasma membrane, which increased the permeability of the cells to exogenous molecules. This "filtroporation" was indicated by uptake of fluorescent dextran molecules up to 500,000 MW in cells deemed viable by trypan blue dye exclusion. The macromolecular uptake was increased if the driving pressure was increased at constant micropore size, or if the micropore size was decreased at constant driving pressure. Larger membrane perturbations permitted uptake of a luciferase reporter plasmid, which resulted in transfection of the CHO cells with the surviving cells expressing luciferase activity after 2 days in culture. This simple and general new method of porating cells in suspension may be optimized to incorporate the desired macromolecules while retaining the maximum viability.     

Flow cytometric assessment of cell viability: a multifaceted analysis

Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

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.     

Flow microfluorometric studies of lectin binding to mammalian cells. II. Estimation of the surface density of receptor sites by multiparameter analysis

A flow-system multiparameter cell analyzer that simultaneously measures and processes fluorescence and cell volume signals from single cells was used to study the binding of fluorescein-conjugated Concanavalin A (Con A–F) to the cell surface. Cells reacted with Con A–F were passed through a flow chamber where sensors measured both cell volume and fluorescence of each individual cell. Sensor signals were electronically processed by first converting the cell volume signals to two-thirds power (proportional to surface area) and then forming the fluorescence-to-surface area ratio. These ratios, which were considered as estimates of the surface density of binding sites, were displayed as frequency distribution histograms using a multichannel pulse-height analyzer for various cell populations differing in cell size. Comparisons between cell lines showed characteristic differences in binding site density. Cell cycle dependent changes were not found for CHO cells synchronized by mitotic selection. An important benefit of this analysis method was the ability to quantitate very weak cell surface fluorescence.     

Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture

To investigate the effect of size-excluded fraction of non-animal protein hydrolysate on growth, viability and longevity of Chinese hamster ovary (CHO) cells, several commercially available protein hydrolysates were evaluated as a feed supplement to chemically-defined protein-free suspension culture. Soy protein hydrolysates showed better supporting capability for cell growth and viability than the other types of hydrolysates. Maximal cell growth was not affected greatly by size exclusion of some soy hydrolysates such as bacto soytone and soy hydrolysates. CHO cells supplemented with size-excluded fractions of the two hydrolysates showed viable cell density and viability almost equal to those with their crude hydrolysates, although soy hydrolysates showed a little better performance. This suggested that the size-excluded hydrolysate fractions of some soy hydrolysate might be a potential culture medium additive to achieve better downstream operation in a large-scale production as well as enhanced productivity.     

Separation of CHO cells using hydrocyclones

Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.     

Slashing the timelines: Opting to generate high‐titer clonal lines faster via viability‐based single cell sorting

Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 ? 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry‐based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:198–207, 2016     

An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.     

定点整合人Bcl-2基因的CHO/dhfr-细胞株的建立

构建重组载体质粒pMCEfrt—Bcl-2,利用FIp—In^TM定点重组系统,在CHO—dhfr^-细胞内定点整合人Bcl-2基因,通过Western印迹检测重组细胞Bcl-2蛋白的表达。通过流式细胞仪和DNA Ladder检测在高NH4C1条件下细胞的凋亡情况;用台盼蓝染色检测在无血清IMDM培养基中细胞的活细胞数目和活细胞比例。结果获得了稳定表达Bcl-2基因的细胞株CHO—Bcl-2,该细胞株能高水平表达Bcl-2蛋白。在无血清培养过程中,CHO—Bcl-2细胞比对照细胞保持高约15％的活细胞比例,细胞总数高25％。CHO-Bcl-2在高NH4^＋（50mmol／L）培养条件下具有较低的凋亡水平。建立了能够高表达Bcl-2基因并具有一定的抗凋亡能力的重组CHO／dhfr^-细胞株。     

A permanent cell viability assay using alcian blue.

The alcian blue dye exclusion method for glutaraldehyde-fixed cells has been utilized with "centrifugal cytology" to prepare permanent records of the viability of individual cells present in suspensions. The viability of spleen cell suspensions separated by linear bovine serum albumin density gradient centrifugation has been measured with this method. Combined light and scanning electron microscopy of nonviable and viable cells demonstrated membrane alterations in alcian blue-stained nonviable cells, while viable cells were spherical and displayed uniform surface features.     

Assessment of bacterial viability status by flow cytometry and single cell sorting

Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytometry. It has previously been shown that four physiological states can be distinguished : reproductively viable, metabolically active, intact and permeabilized. Previous sorting experiments have shown that not all intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnover. To circumvent the limitations imposed by active dye extrusion or cell dormancy on viability measurements used to date (e.g. enzyme activity or cell polarization), a fast triple fluorochrome staining procedure has been developed that takes account of these problems. This allows further cellular characterization of intact cells by : active exclusion of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized). Permeabilized cells were identified by propidium iodide (PI) uptake. The method was validated using an electronically programmable single cell sorter (EPICS Elite^®) and aged Salmonella typhimurium cells. Reproductive viability was determined by sorting single cells to their staining pattern directly onto agar plates. Most polarized cells could be recovered as well as a significant fraction of the depolarized cells, demonstrating that depolarization is a sensitive measure of cell damage but a poor indicator of cell death.     

Selection and characterization of Chinese hamster ovary cells resistant to the cytotoxicity of lectins.

Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Phar) phenotype is stable in the absence of selection; its frequency in a sensitive-population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminal alpha-mannose residues is dramatically reduced (less than or equal to 5% of the activity detected in wild-type CHO cells) in several independent PhaR clones. These clones also exhibit (a) a decreased ability to bind [125I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) and Lens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytoxocity of concanavalin A (Con A); (d) an increased ability to bind 125I-Con A; and (e) decreased surface galactose residues - all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistance closes from each of two complementary CHO auxitrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC, or LCA, and 4-5 "lectin-resistance" phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con AR mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A.     

Cytotoxicity of a GalNAc-specific C-type lectin CEL-I toward various cell lines

We found that CEL-I was a potent cytotoxic lectin. MDCK, HeLa, and XC cells were highly sensitive to CEL-I cytotoxicity and killed in a dose-dependent manner, whereas CHO, L929, and RAW264.7 cells were relatively resistant to CEL-I, and no significant toxicity was observed up to 10 microg/ml. Among these cell lines, MDCK cells showed the highest susceptibility to CEL-I cytotoxicity. A binding study using FITC-labeled CEL-I (F-CEL-I) revealed that the amounts of bound F-CEL-I on the sensitive cell lines were evidently greater than those on the resistant cell lines, suggesting that the different susceptibility of the cell lines to CEL-I cytotoxicity is partly explained by different efficiencies of binding of CEL-I to these cell lines. Interestingly, the cytotoxicity of CEL-I toward MDCK cells was more potent than those of other lectins such as WGA, PHA-L, and Con A, even though these lectins were capable of binding to MDCK cells at comparable levels to CEL-I. Since the cytotoxicity of CEL-I was strongly inhibited by GalNAc, the binding to cell surface specific carbohydrates is essential for the CEL-I cytotoxicity. The trypan blue dye exclusion test indicated that CEL-I caused a disorder of plasma membrane integrity as a relatively early event. CEL-I failed to induce the release of carboxyfluorescein (CF) from CF-loaded MDCK cells as seen for pore-forming hemolytic isolectin CEL-III, suggesting that the primary cellular target of CEL-I may be the plasma membrane, but its action mechanism differs from that of CEL-III. Although CEL-I induced dramatic cellular morphological changes in MDCK cells, neither typical apoptotic nuclear morphological changes nor DNA fragmentation was observed in CEL-I-treated MDCK cells even after such cellular changes. Our results demonstrated that CEL-I showed a potent cytotoxic effect, especially on MDCK cells, by causing plasma membrane disorder without induction of apoptosis.     

A Permanent Cell Viability Assay Using Alcian Blue

The alcian blue dye exclusion method for glutaraldehyde-fixed cells has been utilized with “centrifugal cytology” to prepare permanent records of the viability of individual cells present in suspensions. The viability of spleen cell suspensions separated by linear bovine serum albumin density gradient centrifugation has been measured with this method. Combined light and scanning electron microscopy of nonviable and viable cells demonstrated membrane alterations in alcian blue-stained nonviable cells, while viable cells were spherical and displayed uniform surface features.