Preparation of Living Spermatocytes

1. Fill the culture chamber, prepared as indicated in Subheading 3.1.2., with a layer 0.5-1 mm thick of heavy mineral oil. Discard old culture chambers that might have accumulated dust.

2. Anesthetize the flies on the CO2 source surface and sort a young Drosophila male under the dissection microscope (see Note 4).

3. Using clean curved forceps, place the fly inside the oil-containing well in a position close to an edge and dissect the testis directly under the oil using two No. 5 dissection forceps. This will avoid evaporation during dissection and cell preparation.

4. Remove the unwanted fly parts. Clean the testis of adhering fat using small triangles of filter paper and drag them onto an untouched area of the cover slip, pulling them from the end with the straight No. 5 forceps (see Note 5).

5. Dry all of the liquid surrounding the testis sac using the small filter paper triangles. This will allow the released spermatocytes to attach to the glass afterward.

6. Using a sterile needle, cut each testis into two pieces at a position close to the cells of interest.

7. Using the straight No. 5 forceps, drag each half gently over the surface of the cover slip to let the cysts come out and spread over the surface of the glass (see Note 6).

8. Put the preparation under the microscope. The life-span of these cells in culture is limited, so time counts. Do as specified in Subheading 3.3.1.

0 0

Post a comment