scholarly journals AN AUTOMATIC METHOD FOR VIABILITY ASSAY OF CULTURED CELLS

1970 ◽  
Vol 18 (8) ◽  
pp. 581-589 ◽  
Author(s):  
REGINA O'BRIEN ◽  
PHYLLIS GOTTLIEB-ROSENKRANTZ
Keyword(s):  
Light Intensity ◽  
Trypan Blue ◽  
Cultured Cells ◽  
Fluorescein Diacetate ◽  
Live Cells ◽  
Cell Populations ◽  
Automatic Method ◽  
Viability Assay ◽  
Pass Through

An automatic method for viability assay of a population of cells is described. Cell populations are stained with trypan blue (for dead cells) and fluorescein diacetate (for live cells). A rapid cell spectrophotometer measures changes in light intensity which occur when individual cells pass through the photometric field. Tallies of the number of decreases (absorption and scatter by dead cells) and the number of increases (fluorescence by live cells) in light intensity are recorded and viability percentages are calculated. These automatically determined viability percentages are compared with viability percentages determined by visual counting and classifying of cells from the same populations.

Establishment of Fluorescein Diacetate and Ethidium Bromide (FDAEB) Assay for Quality Assessment of Isolated Islets

2000 ◽  
Vol 9 (5) ◽  
pp. 681-686 ◽  
Author(s):  
Masaaki Miyamoto ◽  
Yoshihiko Morimoto ◽  
Yuka Nozawa ◽  
A. N. Balamurugan ◽  
Baoyou Xu ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Quality Assessment ◽  
Islet Transplantation ◽  
Ethidium Bromide ◽  
Isolated Islets ◽  
Fluorescein Diacetate ◽  
Live Cells ◽  
Viability Assay ◽  
Clinical Islet Transplantation ◽  
Light Excitation

One of the most important factors in clinical islet transplantation is isolation of a great number of islets with good viability. According to viability assessments of isolated islets, the static incubation test and the perifusion test of islets, which are used retrospectively, take much time and need various apparatus. But viability assessments of isolated islets for clinical islet transplantation require a simple, rapid, sensitive, and prospective method. We have developed a microfluorometric viability assay for isolated human, porcine, and dog islets of pancreata using fluorescein diacetate and ethidium bromide (FDAEB). Fluorescein diacetate (FDA) causes live cells to fluoresce green under blue light excitation (490 nm) and ethidium bromide (EB) causes dead cells to fluoresce red. In this study, we investigated the applicability of FDAEB staining to quality assessment of isolated islets for clinical use by correlation with the counting method with insulin secretion of islets. Discrimination of living from dead islets by insulin secretion correlated well with viability as determined by FDAEB staining. The proportion of living islets within isolated canine islets, as measured by microfluorometric counting, was found to correlate highly significantly on low-temperature (24°C) culture (R = 0.831, p < 0.001) and on 37°C culture (R = 0.553, p < 0.05) with the insulin contents of the same islets. Therefore, it is possible to differentiate degrees of viability, and a scoring system is described for this purpose. The FDAEB assay prospectively and easily provides a rapid, accurate, and objective measurement of the proportion of living cells and dead cells in isolated islets for clinical islet transplantation.

Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays

2020 ◽  
Vol 17 (1) ◽  
pp. 2-22 ◽  
Author(s):  
Abdel-Baset Halim
Keyword(s):  
Cell Viability ◽  
Data Interpretation ◽  
Positive Control ◽  
Live Cells ◽  
Proof Of Concept ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Current Cell ◽  
Dying Cells ◽  
Differential Permeability

:Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated.:A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.

The Use of the Vitality and Chemotaxis of Human Polymorphonuclear Leukocytes for the In Vitro Estimation of the Acute Toxicity of the First Ten Chemicals from the MEIC List

1993 ◽  
Vol 21 (1) ◽  
pp. 73-80
Author(s):  
Matteo Valentino ◽  
Francesca Monaco ◽  
Maria Antonietta Pizzichini ◽  
Mario Governa
Keyword(s):  
Ethidium Bromide ◽  
Polymorphonuclear Leukocytes ◽  
Rank Correlation ◽  
Fluorescein Diacetate ◽  
Live Cells ◽  
In Vitro Toxicity ◽  
Ic50 Values ◽  
Acute Cytotoxicity ◽  
Human Polymorphonuclear Leukocytes

The acute cytotoxicity of the first ten MEIC chemicals has been estimated by others in various cell lines. In the present investigation, isolated human polymorphonuclear leukocytes (PMN) from ten healthy non-smoking laboratory personnel were used to assess in vitro toxicity of the same chemicals. The cells were treated with different concentrations of the respective chemicals for three hours and their vitality and chemotaxis were tested. Vitality was measured by fluorescence microscopy after the addition of fluorescein diacetate and ethidium bromide. Living cells which took up and hydrolysed fluorescein diacetate, and dead cells, stained by ethidium bromide, were counted and the percentage of live cells was calculated. Locomotion stimulated by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (F-MLP), was measured in blind-well Boyden chambers and a chemotactic index was calculated. The results were mathematically transformed to produce a linear curve, and then fitted by the linear least squares procedure, from which LC50 and IC50 values were obtained by interpolation. All the chemicals decreased the vitality and inhibited the chemotaxis of the PMN. Obviously the chemotactic test was more sensitive than the vitality one. A correlation (r = 0.933) was found between vitality and chemotaxis inhibition. Spearman rank correlation analysis revealed significant correlations between our results and those from in vitro experiments conducted in other laboratories, as well as with data concerning mouse, rat and human lethal doses.

scholarly journals Noninvasive and safe cell viability assay forEuglena gracilisusing natural food pigment

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6636 ◽  
Author(s):  
Kyohei Yamashita ◽  
Koji Yamada ◽  
Kengo Suzuki ◽  
Eiji Tokunaga
Keyword(s):  
Cell Viability ◽  
Euglena Gracilis ◽  
Food Culture ◽  
Live Cells ◽  
Natural Food ◽  
Synthetic Dyes ◽  
Cell Viability Assay ◽  
Natural Pigments ◽  
Viability Assay ◽  
Good Ability

Noninvasive and safe cell viability assay is required in many fields such as regenerative medicine, genetic engineering, single-cell analysis, and microbial food culture. In this case, a safe and inexpensive method which is a small load on cells and the environment is preferable without requiring expensive and space-consuming equipment and a technician to operate. We examined eight typical natural food pigments to findMonascuspigment (MP) or anthocyanin pigment (AP) works as a good viability indicator of dye exclusion test (DET) forEuglena graciliswhich is an edible photosynthetic green microalga. This is the first report using natural food pigments as cell viability assay.Euglena gracilisstained by MP or AP can be visually judged with a bright field microscope. This was spectrally confirmed by scan-free, non-invasive absorbance spectral imagingA(x, y,λ) microscopy of single live cells and principal component analysis (PCA). To confirm the ability of staining dead cells and examine the load on the cells, these two natural pigments were compared with trypan blue (TB) and methylene blue (MP), which are synthetic dyes conventionally used for DET. As a result, MP and AP had as good ability of staining dead cells treated with microwave as TB and MB and showed faster and more uniform staining for dead cells in benzalkonium chloride than them. The growth curve and the ratio of dead cells in the culture showed that the synthetic dyes inhibit the growth ofE. gracilis, but the natural pigments do not. As the cell density increased, however, AP increased the ratio of stained cells, which was prevented by the addition of glucose. MP can stain dead cells in a shorter time than AP, while AP is more stable in color against long-term irradiation of intense light than MP. Due to the low toxicity of these pigments, viability of cells in culture can be monitored with them over a long period.

Effects of medium and hypothermic temperatures on preservation of isolated porcine testis cells

2010 ◽  
Vol 22 (3) ◽  
pp. 523 ◽  
Author(s):  
Yanfei Yang ◽  
Ali Honaramooz
Keyword(s):  
Trypan Blue ◽  
Cell Isolation ◽  
Live Cell ◽  
Live Cells ◽  
Cell Recovery ◽  
Trypan Blue Exclusion ◽  
Culture Studies ◽  
Porcine Testis ◽  
Peritubular Cells

The effects of medium and hypothermic temperatures on testis cells were investigated to develop a strategy for their short-term preservation. Testes from 1-week-old piglets were enzymatically dissociated for cell isolation. In Experiment 1, testis cells were stored at either room (RT) or refrigeration (RG) temperature for 6 days in one of 13 different media. Live cell recovery was assayed daily using trypan blue exclusion. In Experiment 2, three media at RG were selected for immunocytochemical and in vitro culture studies. Live cell recovery was also assayed daily for 6 days using both trypan blue exclusion and a fluorochrome assay kit. For all media tested, significantly or numerically more live cells were maintained at RG than RT. On preservation Day 3 at RG (cell isolation day as Day 0), 20% FBS-Leibovitz resulted in the highest live cell recovery (89.5 ± 1.7%) and DPBS in the lowest (60.3 ± 1.9%). On Day 6 at RG, 20% FBS- Leibovitz also resulted in the best preservation efficiency with 80.9 ± 1.8% of Day 0 live cells recovered. There was no difference in live cell recovery detected by the two viability assays. After preservation, the proportion of gonocytes did not change, whereas that of Sertoli and peritubular cells increased and decreased, respectively. After 6 days of hypothermic preservation, testis cells showed similar culture potential to fresh cells. These results show that testis cells can be preserved for 6 days under hypothermic conditions with a live cell recovery of more than 80% and after-storage viability of 88%.

Microdevice for Delivering Homogeneous Equi-Biaxial Strain to Live Cells Without the Use of Platen Structures

2013 ◽  
Author(s):  
Qian Wang ◽  
Yi Zhao
Keyword(s):  
Cultured Cells ◽  
Flexible Substrate ◽  
Mechanical Strain ◽  
Live Cells ◽  
Biaxial Strain ◽  
Thin Membrane ◽  
Strain Homogeneity ◽  
Homogeneous Strain

Live cells from most of the membranous tissues such as alveoli are subjected to equi-biaxial strain originated from their extracellular environments. To understand the role of equi-biaxial strain in live cells, a number of engineered methods have been developed for applying such mechanical strain to in vitro cultured cells. Among these methods, deforming a flexible substrate on a circular platen has been widely used [1], and has been miniaturized into millimeter scale for parallel stretching assay (Figure 1a). Nonetheless, the strain homogeneity becomes increasingly challenging at smaller scale, since it requires an ultra-thin membrane, an indentation platen with well controlled dimension, and the highly precise alignment. This obviously increases the fabrication and operation complexities. Devices that deliver homogeneous strain with minimal fabrication and assembling complexities are needed.

scholarly journals Bovine endometrium-derived cultured cells are suitable for lipofection

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mai Shiokawa ◽  
Ryotaro Miura ◽  
Aki Okubo ◽  
Yujiro Hagita ◽  
Itaru Yoshimura ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Transfection Efficiency ◽  
Cell Populations ◽  
Kidney Cells ◽  
Reporter Assay ◽  
Vimentin Expression ◽  
Bovine Kidney ◽  
Short Intervals ◽  
Veterinary Research

AbstractBovine-derived cultured cells, including Madin-Darby bovine kidney cells, are used worldwide; however, lipofection tend to result in low transfection efficiency, which has impeded the progress of veterinary research. We performed experiments to confirm the lipofection efficiency of bovine-derived cultured cells, to identify cells that suitable for lipofection. Several bovine tissues (endometrium, testis, ear tissue and foetal muscle) were collected, and primary cultured cells were prepared. Lipofection assay showed that only bovine endometrium (BE)-derived cells could be transfected efficiently (50‒70%). BE cells can be divided into at least two types of cell populations (BE-1 and BE-2). The BE-1 cells, which were suitable for lipofection, were obtained by passages at short intervals and were negative for cytokeratin- and positive for vimentin-expression; the BE-2 cells did not have these characteristics and were not suitable for lipofection. Furthermore, the BE-1 cells and artificially immortalised cells of BE-1, iBE-1 cells, were utilised in a reporter assay requiring the introduction of multiple DNAs. Endometrial tissues can be collected from living cows, and BE-1 cells can be obtained easily by controlling passaging timing. The production of BE-1 cells and sharing the methods required to prepare them will contribute to the development of veterinary research.

scholarly journals Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

2002 ◽  
Vol 156 (3) ◽  
pp. 511-518 ◽  
Author(s):  
Pierre Barbero ◽  
Lenka Bittova ◽  
Suzanne R. Pfeffer
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Late Endosome ◽  
Live Cells ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network ◽  
Protein Variants

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.

S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells

2001 ◽  
Vol 359 (2) ◽  
pp. 361-367 ◽  
Author(s):  
Elizabeth J. FOX ◽  
Stephanie C. WRIGHT
Keyword(s):  
Cell Cycle ◽  
Cellular Proliferation ◽  
Cultured Cells ◽  
S Phase ◽  
Proliferating Cells ◽  
Specific Expression ◽  
Related Function ◽  
Erythroleukemia Cells ◽  
A Cell ◽  
Pass Through

The Myc/Max/Mad transcription factor network plays a central role in the control of cellular proliferation, differentiation and apoptosis. In order to elucidate the biological function of Mad3, we have analysed the precise temporal patterns of Mad3 mRNA expression during the cell cycle and differentiation in cultured cells. We show that Mad3 is induced at the G1/S transition in proliferating cells; expression persists throughout S-phase, and then declines as cells pass through G2 and mitosis. The expression pattern of Mad3 is coincident with that of Cdc2 throughout the cell cycle. In contrast, the expression of Mad3 during differentiation of cultured mouse erythroleukemia cells shows two transient peaks of induction. The first of these occurs at the onset of differentiation, and does not correlate with the S-phase of the cell cycle, whereas the second is coincident with the S-phase burst that precedes the terminal stages of differentiation. Our results therefore suggest that Mad3 serves a cell-cycle-related function in both proliferating and differentiating cells, and that it may also have a distinct role at various stages of differentiation.

Post-thaw cellular therapy products trypan blue viability assay standardization by dextran40 sample dilution and time-lapse digital microphotography

Cytotherapy ◽  
2015 ◽  
Vol 17 (6) ◽  
pp. S26
Author(s):  
Hadassa Holzapfel ◽  
Domenica Imperato ◽  
Pearl Bongolan ◽  
Anesa Mirza ◽  
Carlo Palesi ◽  
...  
Keyword(s):  
Trypan Blue ◽  
Cellular Therapy ◽  
Time Lapse ◽  
Viability Assay ◽  
Sample Dilution
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