1.0 INTRODUCTION:
Cell viability analysis is a useful tool in various experimental procedures, including those for tumor susceptibility, microbiological resistance, and spontaneous cell death after submission to different experimental conditions. It has been established that cell membrane integrity is a basic criterion for distinguishing dead from live cells. Thus, dyes capable of selectively penetrating the cytoplasm of dead cells have been widely used as vital dyes. The trypan blue (TB) method is a very common assay for evaluating cytotoxicity in experimental investigations where dead cells absorb TB into the cytoplasm because of loss of membrane selectivity, whereas live cells remain unstained. Thus, the relative number of dead and live cells is obtained by optical microscopy by counting the number of stained (dead) and unstained (live) cells using a Neubauer chamber.
2.0 METHODOLOGY:
· Cell culture
· ARPE-19 cells will be obtained from ATCC (cell line CRL-2302; ATCC, LGC Standards GmbH, Wesel, Germany) and cultured from suitable media supplemented with FBS or FCS or as applicable.
· The cells will be maintained at 37°C under 5% CO2 and 100% humidity in DMEM and supplemented with 10% fetal calf serum and antibiotics (200 μl/ml penicillin G, 200μg/ml streptomycin, and 2μg/ml fungizone) and harvested during the logarithmic growth phase.
· Assay
· Reagent Preparation
· 0.4% trypan blue stain and phosphate-buffered saline (PBS) or serum-free medium will be obtained.
· Trypan blue stain will be stored in dark and filtered after prolonged storage.
· As trypan blue stain binds to serum proteins and causes misleading results, the serum-free medium will be used to obtain reliable results.
· Method
· Aliquot of cell suspension being tested for viability will be centrifuged for 5 min at 100 Å~ g and discard supernatant (The size of the aliquot depends on the approximate number of cells present.).
· The aliquot will be contained a convenient number of cells to count in a hemacytometer when suspended in 1ml PBS and then diluted again by mixing with 0.4% trypan blue.
· Resuspend the cell pellet in 1ml PBS or serum-free complete medium as serum proteins stain with trypan blue and can produce misleading results so determinations must be made in serum-free solution.
· Mix 1 part of 0.4% trypan blue and 1 part cell suspension and allow the mixture to incubate ~3 min at room temperature.
· Cells will be counted within 3 to 5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and reduced viability counts.
· Higher concentration of Trypan Blue will be used, but this requires some preliminary testing under the conditions being used to determine if it yields better results.
· Mixing will be performed in a well of a microtiter plate or a small plastic tube using 10 to 20μl each of cell suspension and trypan blue.
· A drop of trypan blue/cell mixture will be applied to a hemacytometer and place the hemacytometer on the stage of a binocular microscope and focus on the cells.
· The unstained (viable) and stained (nonviable) cells will be counted separately in the hemacytometer.
· The total number of viable cells will be multiplied by 2 (the dilution factor for trypan blue) to obtain the total number of viable cells per ml of aliquot.
· The total number of viable and nonviable cells will be added up and multiplied by 2 to obtain the total number of cells per ml of aliquot.
· Calculate the percentage of viable cells as follows:
Viable cells % = the Total number of viable cells per ml of aliquot/Total number of cells per ml of aliquot × 100.
1.0 ENDPOINT PARAMETER(S):
· % Cell viability
2.0 REFERENCE(S):
2.1 Doaa Awad; Imke Schrader; Melinda Bartok; Andreas Mohr; Detlef Gabel Comparative Toxicology of Trypan Blue, Brilliant Blue G, and Their Combination Together with Polyethylene Glycol on Human Pigment Epithelial Cells. Investigative Ophthalmology & Visual Science June 2011, Vol.52, 4085-4090. doi:https://doi.org/10.1167/iovs.10-6336.
2.2 Warren Strober. Trypan Blue Exclusion Test of Cell Viability. Curr Protoc Immunol. ; 111: A3.B.1–A3.B.3. doi:10.1002/0471142735.ima03bs111.
2.3 Özlem Sultan Aslantürk. In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages. http://dx.doi.org/10.5772/intechopen.71923.
2.4 B.A. Avelar-Freitas, V.G. Almeida, M.C.X. Pinto, F.A.G. Moura, A.R. Massensini, O.A. Martins-Filho, E. Rocha-Vieira and G.E.A. Brito-Melo. Trypan blue exclusion assay by flow cytometry. Brazilian Journal of Medical and Biological Research (2014) 47(4): 307-315, http://dx.doi.org/10.1590/1414-431X20143437.
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