Comparison of microplate- and bottle-based methods to age yeast for chronological life span assays

Identification of Tropical Plant Extracts That Extend Yeast Chronological Life Span

“…To quantitate the cell survival upon stress exposure, we also performed an outgrowth kinetics assay (Figure 3, right panels). We previously demonstrated that cell number was directly related to absorbance (OD600) [27]. In the non-stress treated, M. esculenta leaf extract extended CLS in budding yeast throughout the ageing period agreeing with dosedependent result, however the W. bifurcata extract did not extend CLS beyond day 6.…”

“…The CLS assay for screening PEs was based on a 96-well plate CLS assay we had established previously [27]. First, the yeast strain 1783 was streaked on YPD agar and incubated for 2 days.…”

“…In the secondary and confirmatory screens, the outgrowths of yeast cultures in the new 96-well microplate were monitored by measuring the absorbance (A 600 ) every 30 min until 24 h using Bio Microplate Reader HiTS (Cosmo Bio, Tokyo, Japan). The cultures in the 96-well microplate were incubated at 30 • C, with continuous shaking at 210 rpm in the reader [27]. The PEs that increased the survival percentage of cultures throughout the ageing period in the secondary screen (Supplementary Figure S9) were selected for the confirmatory screen.…”

“…An amount of 500 µL overnight culture was centrifuged for 2 min at 10,000 rpm, washed twice and resuspended in 500 µL SC medium. An aliquot of 10 µL cell suspension was transferred to 988 µL SC medium containing 2 µL PEs (M. esculenta, W. bifurcata, T. divaricata leaf extracts) at different final concentrations of 0.1, 1, 5, 10, 50, 100, 200 µg/mL for each PE in a Bijou bottle [27]. As a control, 10 µL cell suspension was transferred to 988 µL SC medium containing 2 µL DMSO in a Bijou bottle.…”

“…To determine the effects of the PEs on yeast CLS, we used an adapted chronological life span assay for 96-well plate screening as previously reported [27]. Briefly, the yeast cells were treated with PEs at a final concentration of 100 µg/mL and were grown and aged in 96-well microplates containing SC media.…”