LABORATORY PROCEDURES FOR PLANT CELL LINES
REFERENCE NO.: PC/1998/2/3.4
TITLE: VIABILITY TEST WITH 2,3,5-TRIPHENYLTETRAZOLIUM CHLORIDE (TTC)
The test is usually performed when a new cell line arrives at the collection centre or when a cell line is recovered from freeze preservation. The test gives a quantitative measure of viability but only in comparison with a reference culture. A reference culture is not available if a new culture is tested, therefore in this case a defined minimum level is used to decide whether a cell line is viable or not. The test is performed either with callus or suspension material.
1. 50 mg of callus material and settled or filtered suspension cells (fresh weight) respectively are weighed into a 15 ml test tube
2. 300 ml of TTC solution (2 % of TTC in TRIS-HCl buffer 0.05 M, pH 7,5) and 1,2 ml TRIS-HCl buffer (0.05 M, pH 7,5) are added to the cells.
3. The suspension is incubated for 6 hours in the dark.
4. After gentle centrifugation the reaction mixture is removed from the cells.
5. 3 ml of ethanol are added to the cells and the suspension is incubated at room temperature overnight.
6. After gentle centrifugation 2 ml of the cell-free supernatant is pipetted into a glass tube with a screw cap.
7. The extinction of the supernatant is measured at 500 nm
8.1. In case of a new accession: If the E500 value is less than 0.05 the cell line is considered to be non viable and the depositor is informed and asked to provide new material (PC/1998/2/3 Appendix 3). If the E500 value is between 0,05 and 0,15 the depositor is informed about the low viability and asked to provide new material or more information (PC/1998/2/3 Appendix 2). To check growth the culture is cultivated for 4 weeks in the case of a callus and for 2 weeks in the case of a suspension. If the E500 value is higher than 0,15 the culture is considered to be viable and cultivated for 4 weeks in case of a callus culture and 2 weeks case of a suspension culture to check growth.
8.2. In case of a cell line recovered from freeze preservation: A comparison is made either with an actual control culture or with recorded values of a control culture. The viability is expressed as a part of the 100 % viability of the control culture.
Guidelines prepared for CABRI by DSMZ, 20 Jan. 1998
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