Data analysis

ROS sensor methods and data analysis – detailed protocol from PhD thesis by Amrita Mukherjee

Staging and dissection for imaging the mitochondrially targeted ratiometric hydrogen peroxide sensor

Flies were allowed to lay eggs on apple-juice based agar medium overnight at 25 ˚C. The agar plate with eggs was then removed and a dollop of yeast paste mixed with bromophenol blue (Sigma-Aldrich) was added; for controls yeast paste only or, for experimentals, yeast paste supplemented with 10mM or 30mM diethyl maleate (DEM; Sigma Aldrich). Eggs were then allowed to develop and larvae to hatch at 25 ˚C. For UAS-dTrpA1 based experiments, larvae were reared at one of the following four temperatures: 23 ˚C, 25 ˚C, 27 ˚C and 29 ˚C. Larvae were then incubated till their guts began to clear of blue food. This was an indication of larvae having stopped feeding and having reached the wandering third instar stage (~100 hours ALH). Thus, staged larvae were then selected for the correct genotype and dissected in 1X PBS saline containing 20 mM N-ethyl maleimide (NEM; pH 7.4), to preserve the oxidation state and to test sensor responses in muscles and neurons. Post dissection, larvae were incubated in 20 mM NEM for 5 minutes followed by fixation in 4% formaldehyde containing 20 mM NEM for 8 minutes at room temperature. Dissected larvae were then equilibrated in 70% glycerol for 30 minutes, mounted in 70% glycerol and imaged immediately. For calibration of sensor response and comparison with published data, imaginal discs were dissected from larvae, fixed as detailed above and immediately mounted in glycerol for imaging. 

Imaging ratiometric hydrogen peroxide sensors 

Dissected larval preparations were imaged on a Leica SP5 Upright DM6000 confocal microscope with a 63x/1.3 N.A. glycerol objective (Leica). Imaginal discs were imaged with a 20x/0.75 N.A glycerol objective. The sensors were excited sequentially at 405 nm and 488 nm (line by line) and the emission was detected between 500-570 nm (muscles), 500-600 nm (imaginal discs) and 500-535 nm (aCC and RP2 motorneurons). Images were saved as 16-bit files. 

Image Processing of ratiometric hydrogen peroxide sensors

ImageJ (National Institutes of Health) was used for processing all images. Prior to analysis, images were corrected for chromatic aberration. For images of imaginal discs, background was subtracted using the rolling ball procedure set to 50 pixels. Images were then converted to the 32-bit format. The intensity of the 488 nm image was thresholded and values below threshold were set to ‘not a number’ (NaN). A ratio image was created by dividing the 405 nm image by the 488 nm image, pixel by pixel, and displayed using the lookup table ‘Fire’. For images of muscles, z-stacks were converted to the 32-bit format and a ratio image created by dividing the 405 nm image by the 488 nm image, pixel by pixel. For images of neurons, the images were converted to the 32-bit format, intensities of the 488 nm image thresholded, with values below threshold set to ‘not a number’ (NaN) and a ratio image created by dividing the 405 nm image by the 488 nm image, pixel by pixel

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