Computational protocol: The Evaluation of Geroprotective Effects of Selected Flavonoids in Drosophila melanogaster and Caenorhabditis elegans

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Protocol publication

[…] In this experiment, two C. elegans strains were used; the Bristol strain N2, kindly provided by Yelena Budovskaya (University of Amsterdam), and the TG38 aak-2(gt33) strain, kindly provided by Alexander Mironov (Engelhardt Institute of Molecular Biology, Moscow). The experiments were performed in liquid culture at 20°C according to a slightly modified version of the protocol set forth by . In brief, worms were synchronized with a bleach solution. After hatching, they were placed in 96-well plates with an S-complete solution and Escherichia coli OP50 as a food source. To prevent the production of progeny, a 0.12 mM solution of 5-fluoro-2′-deoxyuridine (FUDR) (Sigma) was added to C. elegans approximately 45 h after adding the bacteria to L1 worms. Amphotericin B was used at a final concentration of 0.1 μg/mL. To prevent starvation, 5 μl of OP50 (2 × 109 CFU/ml) was added to each well on the 5th, 12th, and 19th day of the worms’ adulthood. The DMSO stock solutions of luteolin (Sigma), chrysin (Acros Organics) and naringin (Acros Organics) were added to N2 nematodes at final concentrations 0.5, 1, 5, 10, and 100 μM. The experiments on the aak-2(gt33) mutant strain were performed using the above concentrations of flavonoids, demonstrating the effect of flavonoids on wild-type worms. This mutant strain has a deletion in the aak2 gene coding, leaving one out of two alpha catalytic subunits of AMPK (). For each experimental group in each experiment 45–110 nematodes were used with the total amount of worms in all performed experiments being no less than 170 for N2 Bristol strain and 110 for TG38 aak-2(gt33) strain. The number of dead worms was counted three times per week. Wells with less than five or more than 15 worms were eliminated to control for the effects of varied food availability. Vibratory perturbation and light exposure were used to induce movement and improve the accuracy of the count. It should be mentioned that in some cases, sediment was observed. The experiment was replicated three times. Some flavonoid concentrations were tested only twice. The statistical analysis was performed for each experiment and for the combined data of all experiments. The survival functions were counted using the Kaplan–Meier method and plotted as survival curves (). The mean, median, minimum and maximum lifespans as well as the age of 90% mortality were calculated. The statistical significance of differences in survival data was estimated using the non-parametric Kolmogorov–Smirnov (), Cox–Mantel () and Gehan-Breslow-Wilcoxon () tests. The statistical significance of differences in maximum lifespan (the age of 90% mortality) was calculated using Wang–Allison test (). The aging process was also assessed by calculating age-dependent (α) and initial (R0) mortality rates in the Gompertz equation (μ(x) = R0 eαx) and mortality rate doubling time (MRDT) using the formula MRDT = ln2/α (). All calculations were performed using Statistica 6.1 (StatSoft, United States) and software environment for statistical computing and graphics R 2.15.1. The effects were considered to be significant if they were observed in at least two out of three experiments in addition to the data from the three experiments combined. [...] In this experiment, the D. melanogaster transgenic line with GstD1-GFP reporter was used. The line was kindly provided by Dr. Tower (University of Southern California, Los Angeles, CA, United States).To measure GstD1-GFP reporter expression, flies were anesthetized and photographed using a fluorescent microscope “MICMED-2 var.11” (LOMO, Russia) and video systems based on the Olympus C7070 digital camera (Olympus, Japan) on the 10th and 30th days of flavonoid consumption. The corrected total cell fluorescence (CTCF) was calculated using ImageJ software (National Institutes of Health, United States) as described elsewhere (). The same assay was performed on flies treated for 12 h with 20 mM paraquat (Methyl Viologen, Sigma) in 5% sucrose (0.2 mL per vial). For each experimental group 10 flies were used. […]

Pipeline specifications

Software tools Statistica, ImageJ
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Drosophila melanogaster, Caenorhabditis elegans, Homo sapiens
Chemicals Ampicillin, Flavonoids, Sirolimus, Luteolin