Measurement of Plant Cell Viability

In cell suspension culture, by measuring the viability of cultured cells and cell clusters, we can effectively understand the growth and viability of cells. Before protoplast culture, it is often necessary to measure the activity of protoplasts to understand the quality of the prepared protoplasts.

Principle

The methods used for the determination of cell (or protoplast) viability include: fluorescein diacetate (FDA) assay, triphenyltetrazolium oxygen reduction assay, Evans blue staining assay, neutral red (NR) assay, MTT assay, XTT method, trypan blue (TB) assay, etc. Hemocytometers were used to observe under a microscope, and the cell viability was expressed as the percentage of the number of living cells in the total number of observations.

Procedures

• Fluorescein Diacetate Method

FDA itself is non-fluorescent and non-polar, and can freely penetrate the plasma membrane into the interior of the cell. After entering the cell, it is hydrolyzed by esterase in living cells to produce a fluorescent polar substance - fluorescein, which cannot freely enter and exit the plasma membrane. Therefore, under a fluorescence microscope, cells with fluorescence can be observed, indicating that the cell is an active cell. Conversely, cells that do not exhibit fluorescence are nonviable cells. The specific operation steps are as follows:

1. Draw 0.5 mL of the prepared cell (or protoplast) suspension into a 10 mm X 100 mm small test tube, and add 0.5 mL of 0.02% FDA solution. Make the final concentration of FDA reach 0.01%. Mix well and incubate at room temperature for 5 min.
2. Fluorescent microscope observation, the excitation filter is QB24, and the suppression filter is TB. The cells that emit green fluorescence are observed as viable cells, and the cells that do not produce fluorescence are inactive dead cells.
3. Viability statistics, cell viability is expressed by the percentage of viable cells in the total number of observed cells.

Cell Viability = Number of Living Cells / Total Number of Observed Cells x 100%

Note: Cells containing chlorophyll may emit yellow-green fluorescence instead of green fluorescence due to the interference of chlorophyll. Non-viable cells emit red fluorescence.

• MTT Assay

Viable cells (or protoplasts) can be used to determine the viability of cells because the dehydrogenase in the cells reduces the light yellow MTT to the blue-purple compound formazane. MTT has good solubility in water, while formazane is insoluble in water, but easily soluble in organic solvents such as ethanol, isopropanol, and dimethyl sulfoxide (DMSO). The absorbance value can be measured by a porous plate spectrophotometer (microplate reader) according to the color depth. Since the amount of formazane produced is directly proportional to the number of living cells reacting, the absorbance value can reflect the number and degree of activity of living cells.

1. Material preparation: prepare suspension (single) cell suspension (or prepare protoplast suspension).
2. Preparation of MTT solution: MTT was prepared into a 0.5 mg/mL solution with pH 7.2, 0.2 mol/L phosphate buffer (PBS).
3. Take 0.5 mL of cell (or protoplast) suspension in a test tube, then add 0.5 mL of MTT solution, and react for 10 min at room temperature.
4. Observe the cell suspension under a microscope, count the number of blue-purple cells, and calculate the cell viability according to the following formula.

Cell Viability = Number of Cells Showing Blue-Purple / Total Number of Observed Cells x 100%

• 2,3,4-Triphenyl Tetrazolium Chloride Reduction Assay

Viable cells (or protoplasts) reduce 2,3,4-triphenyl tetrazolium chloride (TTC) to red due to the activity of oxidoreductase, and the viability of the cells can be determined accordingly. Generally, the number of red cells in the field of view can be observed under a microscope, and the percentage of living cells can be calculated. The red substance can also be extracted with ethyl acetate, and the light absorption value can be measured at 520 nm with a spectrophotometer to calculate the relative viability of the cells.

1. Material preparation: prepare suspension (single) cell suspension (or prepare protoplast suspension).
2. Preparation of TTC solution: TTC was prepared into a 0.001% solution with distilled water.
3. Take 0.5 mL of cell (or protoplast) suspension in a test tube. Then add 0.5 mL of TTC solution and react for 5 min at room temperature.
4. Observe the cell suspension under a microscope, count the number of red cells, and calculate the cell viability according to the following formula.

Cell Viability = Number of Red Cells / Total Number of Observed Cells X 100%

• Staining Assay

Viable cells (or protoplasts) have the characteristic of selectively absorbing foreign substances. When treated with a stain, living cells reject the entry of the stain and therefore do not acquire the color. Dead cells can absorb a large amount of dye and become stained. By counting the number of unstained cells, its viability can be calculated. The specific operation steps are as follows:

1. Material preparation: prepare suspension (single) cell suspension (or prepare protoplast suspension).
2. Prepare dyeing agents, such as magenta, methylene blue, Evans blue, etc., and choose one of them to prepare a concentration of 0.005%~0.01%.
3. Add the dye to the material for dyeing, and it can be observed and counted after a few minutes. Note that the staining time should not be too long, otherwise the viable cells will also be stained with some color, which will affect the accuracy of statistics.
4. To count the unstained cells, the cell viability can be calculated according to the following formula.

Cell Viability = Number of Unstained Cells / Total Number of Observed Cells X 100%

Note:

The method for viability determination of plant cells can also be used for viability determination of protoplasts. For the determination of cell or protoplast viability, one method can be selected, or several viability determination methods can be used at the same time, and then the results can be compared.

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