Evaluation of a Continuous Quantification Method of Apoptosis and Necrosis in Tissue Cultures

In tissue-engineering and other life sciences, there is a growing need for real-time, non-destructive information on apoptosis and necrosis in 2D and 3D tissue cultures. Previously, propidium iodide was applied as a fluorescent marker for monitoring necrosis. In the current study this technique was extended with a fluorescent apoptosis marker, YO-PRO-1, to discriminate between both stages of cell death. The main goal was to evaluate the performance of YO-PRO-1 and propidium iodide during monitoring periods of up to 3 days. Apoptosis was induced in C2C12 cultures and the numbers of YP-positive and PI-positive nuclei were counted in time. The performance of the dual staining was evaluated with a metabolic measure and a probe intensity study. Cell metabolism was unaffected during the first 24 h of testing. In conclusion, the YP/PI dual staining method was found to be a powerful tool in obtaining real-time spatial information on viability in cell and tissue culture without culture disruption.     

Sperm maturation screening and the effect of ecdysone on sperm development of silkworm Bombyx mori

There are two sperm morphs of silkworm, the nucleated spermatozoa (eupyrene) and anucleated spermatozoa (apyrene). Eupyrene sperm cannot complete fertilization successfully without the apyrene sperm. Here a modified rapid and efficient method for sperm identification was developed, after 10 s of fixation in paraformaldehyde and 30 s of 4′6-diamidino-2-phenylindole (DAPI) or propidium Iodide (PI) staining, the sperm bundles can be detected easily using a fluorescence microscope. Sperm maturation process of silkworm from the fifth instar larvae to the adult was described with the above method, the precise time of earliest elongate apyrene bundles was detected on day 2 of pre-pupation, with a ratio of 5% in total sperm bundles, after which the percentage of apyrene sperm bundles increased rapidly and attained a relatively stable ratio of 75% at the end of pupation and nearly 80% after eclosion. Delayed mating leads to apyrene sperm accumulation and damaged fertilization. Previous study showed that ecdysone can increase the frequency of apyrene sperm bundles in vitro. Here 20-hydroxyecdysone (20E) was injected into hemolymph of the 2-d-old fifth instar larvae, the worms entered into mounting period after three days injection, but no apyrene sperm bundles were induced unless day 2 of pre-pupation. Interestingly, maturation of eupyrene sperm bundles were accelerated, and the ratio of eupyrene sperm bundles increased and exhibited a dose-dependent effect after 20E injection, which indicated that the development of eupyrene sperm can be accelerated by ecdysone before pupation of silkworm in vivo. These results will provide new clues for lepidopteran pest control.