A rapid and reliable flow cytometric method for determining Hsp70 levels in tobacco protoplasts

Current methods to determine heat shock protein (Hsp) synthesis or accumulation in plant cells, such as Western blotting and biomet abolic labelling are either indirectly quantitative, labour-intensive or biohazardous. An optimal flow cytometric protocol was developed to measure the intracellular Hsp70/Hsc70 levels in tobacco protoplasts. After heat treatments, protoplasts were fixed in 2% paraformal dehyde-phosphate-buffered saline and dehydrated overnight in methyl cellusolve^TM, followed by permeabilization with Triton X-100 (0. 1% in Protoplast Wash Fluid). Immunolabelling of Hsp70/Hsc70 was done for 1 hour with a mouse monoclonal antibody and detected by;R-Phycoerythrin-conjugated goat anti-mouse IgG using flow cytometry. Flow cytometry detected a significant 1. 2-fold increase in Hsp70/Hsc70 accumulation (P < 0. 001) in protoplasts, while Western blotting, quantified by image anal ysis, showed induction under similar conditions but at lower significance (P < 0. 05). The coefficients of variance for flow cytometry and Western blotting were 30. 7 and 49. 8 respectively. Optimum temperature of heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts occurred at 40 °C. Flow cytometry is proposed as a quantitative, more reproducible and rapid alternative to Western blotting for the detection of Hsp70 accumulation in plant cells.