Evaluation of cell viability Coffea arabica – Evans blue Coffea arabica – MTT essay Solanum lycopersicum – FDA essay Percentage of viable cells Cell viability tests allow the discrimination between live and dead cells The percentage of viable cells (%V) is determined with the following formula Common reagents to evaluate the percentage of cell viability Several tests can be performed to evaluate the number of viable cells within a cell population: 1. Fluorescein diacetate (FDA) FDA 2. Triphenyltetrazolium chloride (TTC) TTC FDA reveals viable cells with a green fluorescent signal Cytoplasmic esterases Fluorescein diacetate (FDA) Non-fluorescent Lipophilic ↓ It easily crosses biological membranes Fluorescein Fluorescent Non-lipophilic ↓ It cannot cross biological membranes FDA test can be used to assess the viability of in vitro cultivated cells, protoplasts and cells belonging to a tissue MTT viability test TTC is a redox indicator that is used in biochemical experiments to detect cellular respiration Oxidized form ↓ Water-soluble White color Reduced form ↓ Insoluble in water Red color A Orchis provincialis B C D E Viability tests with TTC (A, B, E) and with FDA (C, D), performed on seeds of the Orchis provincialis species. (A) non-viable embryo, treated with TTC; (B) viable embryo, treated with TTC; (C) seed with viable embryo, treated with FDA and observed in bright field; (D) same seed as C, observed under blue light; (E) seeds with viable embryo (left) and with non-viable embryo (right), both treated with TTC. Trypan blue exclusion test of tell viability Cells are suspended in PBS containing trypan blue and then examined to determine the percentage of cells that have clear cytoplasm (viable cells) versus cells that have blue cytoplasm (nonviable cells) Live cells possess intact cell membranes that exclude trypan blue Live cell Dead cell Growth parameters Cell viability •FDA •TTC Growth curve Evaluation of fresh weight and dry weight Pakage cell volume (PCV) Volume of suspended cells Supernatant (culture medium) Pellet (cell biomass) Quantification of cell growth Cultured cells (calli or suspended cells) Time Growth • Increase in cell number • Increase in biomass weight • Increase in biomass volume The main parameters that are considered to measure cell growth are: - Number of cells - Fresh weight - Dry weight - Packed cell volume Example 1: Quantification of the increase in fresh callus biomass • Inoculate in 10 petri dishes about 2 g of callus • After 25 days weigh the plates with the grown callus • Transfer callus to 10 plates with renewed soil • Weigh the empty plates and for difference with the floods evaluate the growth of the callus • Repeat after 25 days (subcultures) In this way it is possible to compare the impact of different media or different culture conditions on cell growth. Example 2: Quantification of the increase in dry callus biomass In this case the sample must be sacrificed!!! • Prepare 20 Petri dishes, each containing 2 g of callus •Collect the callus from 3 plates, dry at 60°C until it reaches a constant weight (initial dry weight) •After 25 days, take the callus from 3 plates, dry in the oven as described above and weigh (final dry weight) •The increase in dry weight will be given by the difference between the final and initial dry weight The dried cellular biomass could be used for the analysis of the metabolites produced Example 3: Determination of the increase in cell density by optical microscopy (cell counting) Applicable to suspension cultures of cells and protoplasts in liquid medium Cell density = number of cells per unit volume • Prepare a cell suspension consisting of single cells or small clumps in active division • After a certain period of time, take an aliquot and separate the cells by chemical (chromium tetroxide) or enzymatic (pectinase) treatment • Carry out the count, using an hemocytometer (Thoma chamber, Burker chamber) Usage of Thoma cell counting chamber Thoma chamber contains 1 large central square (1x1 mm) which can be completely seen with 10X objective. This square is divided into 16 medium squares that can see completely with 40X objective. Each medium square is divided in 25 small squares (9 of them are divided in half) . When the sample is placed under the coverslip, the cell suspension reaches a height of 0.1 mm, then the volume is: 1 x 1 x 0,1 = 0,13 mm = 10-4 ml With the 10X objective the counting area must be located. To count the cells, the 40X objective must be used. All the cells in the 16 medium squares must be counted according to the following criteria: ………………………………… All the cells within each medium square and those that are over the top and right sides of the square (even when they are partially out) are counted. Following this approach, in the figure the cells in green will be counted, but not the cells in red. If we have counted N cells in one of the large squares (that is, in 16 medium squares), the concentration of our sample will be: N x 104 cell/ml When prior to counting we concentrated or diluted the initial sample, we must take into account the concentration-dilution factor (f): N x 104 x f cel/ml initial cell suspension = diluted cell suspension x concentration-dilution factor Example 4: Determination of the increase in packed biomass volume (PCV) Applicable to suspension cultures of cells and protoplasts in liquid medium • Transfer a known volume of a cell suspension into a Falcon tube • Centrifuge at 1,000 rpm for 3-5 min • Read the volume occupied by the packed cells on the total volume • Make multiple replicas
