2.8. Rapid Iterative Negative Geotaxis (RING) Assay (Climbing Assay)

The RING assay is based on gravity reversal reaction when there is a force acting in the same gravity direction on fly [29]. More healthy flies are able to climb higher in the same period of time. This experiment can be performed to test the effect of a factor on the mobility of adult fly. This assay was performed as described previously [30] with some modifications. After being fed extract-containing standard medium, newly eclosed flies were anesthetized with diethyl ether and randomly selected to collect 160–200 male flies. These flies were then divided into 2 groups including short-term treatment group and long-term treatment group. The short-term treatment group was fed on the standard medium, while the long-term treatment group continued to be fed the extract-containing standard medium. The flies were maintained at 28°C and transferred to tubes containing new medium every 2 days. The flies were kept on new medium overnight before carrying out the climbing assay. They were then anesthetized with diethyl ether and separated into climbing vials at a density of 10 flies per vial. These flies had at least 60 minutes of rest before the assay. After that, each vial was tapped to collect the flies to the bottom and the movement of these flies was recorded. This procedure was repeated 8 times. The climbing index was the average percentage of flies which passed the height of 6 cm after 5 seconds. The relative levels of improvements in climbing ability were calculated as follows: climbing index of 11-day-old flies divided by mean of climbing index of 1-day-old flies and then normalized by the mean of nontreated control. Raw data were collected by Microsoft Excel 2016 (Microsoft, USA). The value was calculated, statistically analyzed, and graphed using GraphPad Prism 7.00 (GraphPad Software, USA).