Clear bottom 96 well plate

Ten micrograms of isolated mitochondria were resuspended in 170 µl of 100 mM triethanolamine buffer, pH 8.0, containing 1.2 mM NADP⁺ and 10 µL isocitrate dehydrogenase (Sigma-Aldrich, I2002) in a clear bottom 96-well plate (Falcon). Twenty microliters of cis-aconitate was added (final concentration 240 µM) and reduction of NADP⁺ was monitored at 340 nm for a total of 10 min. The rate of reaction was calculated from the slope of the linear part of the kinetic curve. As a control, activity was also measured in pure buffer or mitochondrial samples before and after the addition of cis-aconitate^{54 (link)}. Aconitase activity was normalized to citrate synthase activity and displayed as a percentage of WT activity.
For citrate synthase activity, 20 µg of isolated mitochondria were resuspended in 100 µl of 10 mM Tris-HCl buffer, pH 7.5, 0.2% Triton X-100 (v/v) and 200 µM of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), in a clear bottom 96-well plate (Falcon). Fifty microliters of acetyl-CoA (2 mM) was added to this solution. After 5 min of incubation, reaction was started by adding 50 µl of oxaloacetate (2 mM) and turn-over of acetyl-CoA was monitored by observing the absorbance at 412 nm for 10 min. The initial rate of reaction was calculated from the slope of the linear part of the kinetic curve^{55 (link)}.

Leaf discs (7 mm) were made with a hole punch and placed on a 0.5% (w/v) agar pad in wells of clear-bottom 96-well plate (Falcon) sealed with Microseal “A” film (Bio-Rad). The film was punctured over each well with a needle to allow for gas exchange. The prepared plates were dark-acclimated for a minimum of 6 h before placement in a BioTek Cytation 3 Imaging Reader. The baseline level of light transmittance through the leaf discs was calculated from measurements of absorbance values of 660-nm red light taken every 2 min for 20 min (red light does not activate chloroplast movement). To induce chloroplast movement in the cells, the plate reader was programmed to eject the plate for exposure to the selected light intensity for 2 min. The plate was then moved back into the plate reader for recording of transmittance values (660-nm red light absorbance) for each well. After each reading, the plate was re-ejected to return to the blue light treatment. The cycle of recording transmittance values and incubating with blue light was repeated for the indicated time periods for a given light treatment. The calculated changes in light transmittance values were normalized to be relative to the starting “dark” position values.

Leaf discs (7 mm) were made with a hole punch and placed on a 0.5% (w/v) agar pad in wells of a clear-bottom 96-well plate (Falcon) sealed with Microseal “A” film (Bio-Rad). The film over each well was punctured with a needle to allow for gas exchange. The prepared plates were dark-acclimated for a minimum of 6 h before placement in a BioTek Cytation 3 Imaging Reader. The baseline level of light transmittance through the leaf discs was calculated from measurements of absorbance values of 660 nm red light taken every 2 min for 20 min (red light does not activate chloroplast movement). To induce chloroplast movement in the cells, the plate reader was programmed to eject the plate for exposure to the selected light intensity for 2 min. The plate was then moved back into the plate reader for a recording of transmittance values (660 nm red light absorbance) for each well. After each reading, the plate was re-ejected to return to the blue-light treatment. The cycle of recording transmittance values and incubating with blue light was repeated for the indicated time periods for given light treatment. The calculated changes in light transmittance values were normalized to be relative to the starting “dark” position values.