2. Take out from the C or CE stock solution the total amount that
will be necessary to perform the whole experiment, place in a
1.5 mL vial and completely dried under nitrogen stream (see
Note 5).
3. Store the dried C or CE aliquot at À20
C until preparing the
NGM dishes supplemented with C or CE (see Note 6).
3.2 Preparation
of NGM Petri Dishes
with E. coli OP50
Supplemented with C
or CE for Feeding
Experiments
We recommend preparing the NGM dishes supplemented with C
or CE in the same day of the feeding experiment.
1. In exploratory experiments we recommend to perform a doseresponse experiment with 5–6 concentrations of C or CE
covering a range from 0.005 μg/mL to 0.1 μg/mL (e.g.,
0.005, 0.01, 0.025, 0.05, 0.1 μg/mL) (see Note 7). The
NGM dish contains a volume of 10 mL, therefore a total
amount of 0.05, 0.1, 0.25, 0.5, and 1 μg of C or CE is added
per dish. To that end, dissolve the dried aliquot in the appropriate volume of chloroform–ethanol (ratio 1:15, v/v) and
prepare the dilutions.
2. The solution containing the corresponding amount C or CE
are directly spread on the agar dish surface in sterile conditions
and spread with a Drigalski spatula. To minimize the variability,
the same volume of C or CE solution will be added to each
dish. In control NGM dishes the same volume of solvent used
for C or CE will be added to the dish (see Note 8).
3. In order to get a comparison of the antioxidant capacity
provided by carotenoids respect to other well-recognized antioxidant compounds, we use vitamin C at a final dose of 0.1 μg/
mL as positive control in the oxidative stress assays. Commercial vitamin C is prepared immediately before the assay and is
added on the agar dish surface.
3.3 Preparation
of Nematode
Synchronized
Population for Feeding
Experiments
All assays should be performed with age-synchronized nematodes
populations without experienced food deprivation.
1. To obtain a synchronized population of nematodes, place
gravid nematodes on 35 mm NGM dishes (control) containing
E. coli OP50 strain or NGM supplemented with the
corresponding C or CE or vitamin C at the corresponding
doses. Allow to lay eggs for 2 h.
2. Remove the adults, and incubate the hatched eggs for 4 days at
20
C to obtain synchronized young adults (see Note 9).
3. Place young synchronized adults on NGM dishes (control),
supplemented NGM dishes with a specific of C or CE concentration or with vitamin C (0.1 μg/mL) containing E. coli OP50
strain. The nematodes are incubated for 7 days at 20
C. During this incubation period the nematodes uptake the
Carotenoids and Oxidative Stress Assay in C. elegans
393
will be necessary to perform the whole experiment, place in a
1.5 mL vial and completely dried under nitrogen stream (see
Note 5).
3. Store the dried C or CE aliquot at À20
C until preparing the
NGM dishes supplemented with C or CE (see Note 6).
3.2 Preparation
of NGM Petri Dishes
with E. coli OP50
Supplemented with C
or CE for Feeding
Experiments
We recommend preparing the NGM dishes supplemented with C
or CE in the same day of the feeding experiment.
1. In exploratory experiments we recommend to perform a doseresponse experiment with 5–6 concentrations of C or CE
covering a range from 0.005 μg/mL to 0.1 μg/mL (e.g.,
0.005, 0.01, 0.025, 0.05, 0.1 μg/mL) (see Note 7). The
NGM dish contains a volume of 10 mL, therefore a total
amount of 0.05, 0.1, 0.25, 0.5, and 1 μg of C or CE is added
per dish. To that end, dissolve the dried aliquot in the appropriate volume of chloroform–ethanol (ratio 1:15, v/v) and
prepare the dilutions.
2. The solution containing the corresponding amount C or CE
are directly spread on the agar dish surface in sterile conditions
and spread with a Drigalski spatula. To minimize the variability,
the same volume of C or CE solution will be added to each
dish. In control NGM dishes the same volume of solvent used
for C or CE will be added to the dish (see Note 8).
3. In order to get a comparison of the antioxidant capacity
provided by carotenoids respect to other well-recognized antioxidant compounds, we use vitamin C at a final dose of 0.1 μg/
mL as positive control in the oxidative stress assays. Commercial vitamin C is prepared immediately before the assay and is
added on the agar dish surface.
3.3 Preparation
of Nematode
Synchronized
Population for Feeding
Experiments
All assays should be performed with age-synchronized nematodes
populations without experienced food deprivation.
1. To obtain a synchronized population of nematodes, place
gravid nematodes on 35 mm NGM dishes (control) containing
E. coli OP50 strain or NGM supplemented with the
corresponding C or CE or vitamin C at the corresponding
doses. Allow to lay eggs for 2 h.
2. Remove the adults, and incubate the hatched eggs for 4 days at
20
C to obtain synchronized young adults (see Note 9).
3. Place young synchronized adults on NGM dishes (control),
supplemented NGM dishes with a specific of C or CE concentration or with vitamin C (0.1 μg/mL) containing E. coli OP50
strain. The nematodes are incubated for 7 days at 20
C. During this incubation period the nematodes uptake the
Carotenoids and Oxidative Stress Assay in C. elegans
393