The overall goal of this procedure is to assess a relative loss of lysosomal acidity in the intestinal lysosomes of C.elegans. This method can help answer key questions in the field of autophagy by indirectly providing information about lysosomal function and protein degradation. The main advantage of this technique is that it is simple, inexpensive, convenient, and reproducible.
We first had the idea for this method when we observed the staining technique being used for assessing vacuolar alkalinization in yeast. Visual demonstration of this method is important because certain steps, such as adding cDCFDA to OP50 food and imaging control two day old wild type worms are critical. After preparing the NGM plates, let the plates dry in room temperature for two days with the lid closed.
Then, inoculate the OP50 bacteria in the sterile Luria broth. After inoculating, leave the inoculant in the incubator until the OD is between 0.2 and 0.4. Then, place a drop of the OP50 inoculant in the center of the dry NGM plate.
Next, use a spreader to spread the inoculant in patch roughly two by two centimeters. Approximately after 15 minutes, invert the OP50 plate after the inoculant has dried up. Then, incubate the plate at 37 degrees Celsius for 36 hours.
Once the incubation is over, transfer the plate at four degrees Celsius til use. Then, prepare a working stock of 10 millimolar cDCFDA in M9 buffer. Store the cDCFDA solution away from light at minus 20 degrees Celsius.
Then, pipette 100 microliters of 10 millimolar cDCFDA to cover the entire OP50 patch uniformly on the plate. Leave the cDCFDA plates in a dark box on the bench top for about 30 minutes. After the cDCFDA has permeated the OP50 patch completely, transfer 20 worms on the plate, then leave the plate in inverted condition for about 14 hours at 20 degrees Celsius.
Next, take two strips of labeling tapes and place them 1.5 inches apart on a smooth, flat mirror surface. Then, coat the surface of the mirror with water repellent spray, and remove the excess liquid out. Next, melt 2%agarose in distilled water in the microwave.
After the agarose has liquified, place a drop of agarose between the two strips of the tapes and cover the drop with the microscope slide. After the agarose has solidified in the shape of a circular pad under the slide, flip the slide. Then, add 10 to 15 microliters of five millimolar sodium azide on the agarose pad.
Now, transfer the worms from the cDCFDA supplemented plate to a clean NGM plate without any OP50. Let the worms move around for a few seconds for the OP50 to be removed from their surface. Then, transfer the worm on the agarose pad containing five millimolar sodium azide.
Quickly cover the agarose pad with a cover slip. Next, use 60X magnification lens and capture images in a single plane of the intestine from two day old wild type and mutant worms. Then, open ImageJ and click on the file.
Then, click Open to open the file that needs to be quantified. After the image has loaded, use the oval-shaped Selection Tool to select the region of interest. Then, click Analyze, and finally, Measure to quantify the fluorescence intensity of the region of interest.
To study the lysosomal functional activity, cDCFDA staining intensity is quantified using confocal imaging. In this image, there is a robust cDCFDA fluorescence signal intensity in the intestinal lysosomes of wild type day two C.Elegans. This indicates a functionally competent acidic lysosome.
On the contrary, the fluorescence intensity is considerably suppressed in the intestinal lysosomes of wild type day eight old worms. This is in comparison to the intestinal lysosomes of day two worms. The result indicated the presence of functionally incompetent alkaline lysosome in day eight old worms.
Interestingly, the cDCFDA fluorescence in intensity is also markedly reduced in both day two and eight old RNAi worms when compared to the wild type worm. Here, the vha gene, encoding the vacuolar ATPase subunits in the lysosomal lining has been knocked down in the day two and day eight old worms. Once mastered, this entire staining and microscopy regimen can be completed in less than 18 hours, if performed properly.
While attempting this procedure, it is important to remember to incubate worms in cDCFDA supplement in OP50 food for at least 14 hours. After watching this video, you should have a good understanding of how to assess loss of lysosomal acidity in the intestinal lysosomes of live C.elegans.
