1. Basal S medium plates are prepared as described previously. The
H 2 O 2 working solution is prepared and added to the medium
at final dose of 2 mM (see Note 11).
2. Nematode adults previously incubated for 7 days at 20
C on
NGM dishes (control), supplemented NGM with a specific of
C or CE concentration or with vitamin C (0.1 μg/mL) (see
step 3 from Subheading 3.2) are transferred to freshly prepared
Basal S medium dishes containing 2 mM H 2 O 2 and left for 5 h.
This incubation with H 2 O 2 provokes an acute oxidative stress.
3. After incubation, the nematodes are scored for viability under
the stereomicroscope. The viability of the nematodes is scored
by touching them with a platinum wire and considering dead
nematodes those that do not longer respond to prodding.
4. Statistically evaluate the viability of nematodes (percentage of
survival) between nematodes cultured in control and treatment
conditions after oxidative stress by means of one-way analysis of
variance (ANOVA) and the Tukey test for comparative analysis.
3.5 Carotenoid
Bioassimilation Assay
To confirm C or CE intake by nematodes, a procedure has been
developed that determines the presence and composition of the
carotenoids in the nematodes upon the feeding experiment by
HPLC-PAD. All solvents use in this section are HPLC grade, unless
is specified.
1. Obtain a synchronized population of C. elegans of 5 days age
using the same procedures described in steps 1 and 2 of Subheading 3.2.
2. Prepare at least 50 dishes of NGM (control) with OP50 and
NGM with OP50 supplemented with C or CE at final concentration of 1 μg/mL (see Note 12).
3. Obtain around 150–200 age-synchronized nematodes per dish
and incubate for 24 h at 20
C.
4. Recover the nematodes from the dish surface by adding 1 mL
of M9 buffer to the dish and gently shake it (see Note 13).
5. Transfer the M9 buffer containing worms from dishes to
50 mL tubes. For each condition (NGM-control and
NGM-carotenoid) pool in one single tube the recovered M9
buffer with the nematodes.
6. Centrifuge the 50 mL NGM-control and NGM-carotenoid
tubes at 700 Â g, 2 min, 20
C. Discard the supernatant.
7. Add 20 mL of M9 buffer to each tube, gently shake, and repeat
step 5. Repeat this step twice.
8. Add 10 mL of M9 buffer to each tube and incubate for 2 h at
20
C to facilitate the removal of gut microbiota from the
nematodes. Discard the supernatant.
Carotenoids and Oxidative Stress Assay in C. elegans
395
H 2 O 2 working solution is prepared and added to the medium
at final dose of 2 mM (see Note 11).
2. Nematode adults previously incubated for 7 days at 20
C on
NGM dishes (control), supplemented NGM with a specific of
C or CE concentration or with vitamin C (0.1 μg/mL) (see
step 3 from Subheading 3.2) are transferred to freshly prepared
Basal S medium dishes containing 2 mM H 2 O 2 and left for 5 h.
This incubation with H 2 O 2 provokes an acute oxidative stress.
3. After incubation, the nematodes are scored for viability under
the stereomicroscope. The viability of the nematodes is scored
by touching them with a platinum wire and considering dead
nematodes those that do not longer respond to prodding.
4. Statistically evaluate the viability of nematodes (percentage of
survival) between nematodes cultured in control and treatment
conditions after oxidative stress by means of one-way analysis of
variance (ANOVA) and the Tukey test for comparative analysis.
3.5 Carotenoid
Bioassimilation Assay
To confirm C or CE intake by nematodes, a procedure has been
developed that determines the presence and composition of the
carotenoids in the nematodes upon the feeding experiment by
HPLC-PAD. All solvents use in this section are HPLC grade, unless
is specified.
1. Obtain a synchronized population of C. elegans of 5 days age
using the same procedures described in steps 1 and 2 of Subheading 3.2.
2. Prepare at least 50 dishes of NGM (control) with OP50 and
NGM with OP50 supplemented with C or CE at final concentration of 1 μg/mL (see Note 12).
3. Obtain around 150–200 age-synchronized nematodes per dish
and incubate for 24 h at 20
C.
4. Recover the nematodes from the dish surface by adding 1 mL
of M9 buffer to the dish and gently shake it (see Note 13).
5. Transfer the M9 buffer containing worms from dishes to
50 mL tubes. For each condition (NGM-control and
NGM-carotenoid) pool in one single tube the recovered M9
buffer with the nematodes.
6. Centrifuge the 50 mL NGM-control and NGM-carotenoid
tubes at 700 Â g, 2 min, 20
C. Discard the supernatant.
7. Add 20 mL of M9 buffer to each tube, gently shake, and repeat
step 5. Repeat this step twice.
8. Add 10 mL of M9 buffer to each tube and incubate for 2 h at
20
C to facilitate the removal of gut microbiota from the
nematodes. Discard the supernatant.
Carotenoids and Oxidative Stress Assay in C. elegans
395