Multi range large particle flow cytometer biosorter

Intracellular ROS content was measured by the 2′,7′-dichlorofluorescein diacetate (H2DCFDA) assay. L3 stage synchronized N2 nematodes were treated or untreated with 100 µg/mL of Mexican marigold petal extract and incubated at 20 °C until the read-out point of the experiment (Young worms: 5 days old; Old worms: 12 days old). In the old population, 15 μg/mL FUDR (Sigma-Aldrich, St. Louis, MO, USA) was applied in order to avoid egg laying during the fertile phase. Furthermore, the animals were moved to new plates with fresh treatment and food twice per week in the case of the aged worms, and once per week for the young group. After that time, all nematodes were washed with M9 medium and incubated for 2 h with 25 μM DCFDA at 20 °C. The fluorescence intensity was determined by a Multi-Range Large Particle Flow Cytometer Biosorter (Union Biometrica, Holliston, MA, USA). At least 300 worms were used per group. The results are expressed as a percentage of the young control using the mean of the yellow fluorescence intensity, which is related to the ROS content.

Mitochondrial ROS content was measured by the Mitotracker Red CM-H2 XRos assay (Thermo Fisher, Waltham, MA, USA). L3 stage synchronized N2 nematodes were treated or untreated with 100 µg/mL of T. erecta extract and incubated at 20 °C until the read-out point of the experiment (Young worms: 5 days old; Old worms: 12 days old). In the old population, 15 μg/mL FUDR (Sigma-Aldrich, St. Louis, MO, USA) was applied in order to avoid egg laying during the fertile phase. Furthermore, the animals were moved to new plates with fresh treatment and food twice per week in the case of the aged worms, and once per week for the young group. After that time, all nematodes were washed with M9 medium and transferred to NGM plates containing 10 μM Mitotracker mixed with dead E. coli OP50 as a food source. Worms were incubated with the dye for 2 h at 20 °C and then fluorescence intensity was measured using a Multi-Range Large Particle Flow Cytometer Biosorter (Union Biometrica, Holliston, MA, USA). At least 300 worms were used per group. The results are expressed as a percentage of the young control using the mean of the red fluorescence intensity, which is related to the mitochondrial ROS content.

N2 synchronized eggs were placed on plates with or without the
treatments (0, 100, 500, and 1000 μg/mL) and incubated for 48
h at 20 °C. After that, all groups were washed with M9 medium
and exposed (except the CTL basal group) to 2.5 mM 2,2′-azobis-2-amidinopropane
dihydrochloride (AAPH) for 15 min. Then, the AAPH was removed by wash
with M9, and worms were incubated for 2 h with 25 μM dichlorodihydrofluorescein
diacetate (DCFDA) at 20 °C. A Multi-Range Large Particle Flow
Cytometer Biosorter (Union Biometrica, Massachusetts, USA) was used
to measure the fluorescence intensity of, at least, 300 worms per
group. ROS content was estimated as a percentage of control of average
yellow fluorescence intensity.