Drosophila serves as good model system for studying mitochondrial function. Here we present a protocol for measuring citrate synthase activity in tissue homogenate from adult flies. This protocol does not require isolation of mitochondria.
It is fast and simple and it is suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues. Begin by collecting adult flies for each sample, making sure to collect at least triplicate samples for each genotype. Prepare 500 microliters of ice-cold extraction buffer with 20 millimolar HEPES, one millimolar EDTA, and 0.1%triton X-100 in a 1.5-milliliter test tube for each sample.
Anesthetize the adult flies with carbon dioxide on an anesthesia pad. To isolate the thoraxes, fix them with a pair of forceps and isolate the fly abdomens by cutting along the border of the thorax and abdomen with a pair of scissors. Collect the fly thoraxes for the citrate synthase activity assay.
Transfer 10 adult fly thoraxes to 100 microliters of ice-cold extraction buffer immediately and homogenize them with a pestle on ice. Keep the samples cold by homogenizing for five to 10 seconds on ice, letting them sit on ice for five seconds, then repeating until all tissues are completely homogenized. Aliquot 10 microliters of each homogenized sample into a new tube for protein content measurement, keeping these tubes on ice as well.
Add 400 microliters of ice-cold extraction buffer to each remaining sample for a total volume of 500 microliters and pipette up and down gently to mix. For each reaction, add one microliter of diluted cell lysate to 150 microliters of the freshly prepared reaction solution. Mix thoroughly by gently pipetting, making sure to avoid bubble formation, then use a plate reader to measure the absorbance at 412 nanometers every 10 to 30 seconds for four minutes at 25 degrees Celsius.
Plot the data as absorbance over time, then calculate the slope for the linear portion of the curve. Finally, divide the reaction rate or slope by the sample protein concentration to normalize the citrate synthase activity. It was hypothesized that knockdown of dRNF34 in Drosophila muscle would increase mitochondrial citrate synthase activity while knockdown of dPGC-1 would reverse this increase.
An initial linear enzymatic rate was established for each assay;the slopes of the trendlines represent the maximal reaction rates which are equivalent to the maximal citrate synthase activities of the different genotypes. The maximal citrate synthase activities were calculated from the kinetic curves and normalized by protein concentration. The results demonstrated that the synthase activity of fly thoraxes indeed increased with muscle-specific dRNF34 knockdown and the increase was reversed by muscle-specific dPGC-1 knockdown.
When homogenizing the sample, it is important to tip the tube and check the homogenate to make sure that there are no clots and that the tissues in the tube are completely homogenized. After sample collection, all sample treatments should be performed on ice. The citrate synthase activity assay is used to quantify intact mitochondrial mass.
Proper quantification of intact mitochondrial mass is very important for evaluating mitochondrial function.
