12. Dry the pooled acetone/dichloromethane extracts for each
condition under nitrogen stream (see Note 6).
13. Dissolve the dried residue obtained for each condition in
250 μL dichloromethane, add 250 μL MilliQ water and vortex
for 30 s. Recover the organic phases in a 1.5 mL vial, reextract
the aqueous phase twice with 250 μL of dichloromethane.
14. Dry the pooled organic phases under a nitrogen stream and
store at À20
C until HPLC-PAD analysis (see Note 6).
15. Analyze the presence of carotenoids in the nematode pellet
extracts by using a HPLC-PAD equipped with a C30 analytical
column (4.6 Â 250 mm, 5 μm) couple to a C30 guard column
(20 Â 4.0 mm, 5 μm). In this procedure it is describe the
chromatographic conditions and equipment that is routinely
used in our laboratory which can resolve most of the common
carotenoids present in fruits or fruit-based food. However,
these conditions can be modified and adapted depending on
the carotenoids composition in the extracts under study.
16. Dissolve the nematode pellet extract in 30 μL of methanol–
acetone–chloroform (2:1:3, v/v/v) and centrifuge at
12,300 Â g, 1 min at 20
C.
17. Inject 20 μL on the HPLC system using the method described
elsewhere [33] with a ternary gradient elution. The initial
solvent composition consisted of 90% methanol (MeOH), 5%
water and 5% methyl tert-butyl ether (MTBE). The solvent
composition changes in a linear fashion to 95% MeOH and 5%
MTBE at 12 min. During the next 8 min the solvent composition changes to 86% MeOH and 14% MTBE. After reaching
this concentration the solvent is gradually changed to 75%
MeOH and 25% MTBE at 30 min. Following 20 min solvent
composition changes linearly being, at 50 min, 50% MeOH
and 50% MTBE. The final composition is reached at 70 min
and consisted of 25% MeOH and 75% MTBE. The initial
conditions are established in 5 min and equilibrated for
15 min before the next injection. The flow rate is 1 mL/min,
and the column temperature is set to 25
C. The PAD is set to
scan from 250 to 540 nm, and for each elution a Maxplot
chromatogram is obtained, which plots each carotenoid peak
at its corresponding maximum absorbance wavelength.
18. Compare chromatograms obtained with the pellet extracts of
nematodes grown on NGM-control and NGM supplemented
with carotenoid, and identify carotenoids analyzing the spectra
of the chromatographic peaks (see Notes 16 and 17).
Figure 2b shows chromatograms obtained from C. elegans
grown in NGM control and NGM supplemented with
β-cryptoxanthin at 1 μg/mL. Only one carotenoid peak
which spectrum and retention time matched with
Carotenoids and Oxidative Stress Assay in C. elegans
397
condition under nitrogen stream (see Note 6).
13. Dissolve the dried residue obtained for each condition in
250 μL dichloromethane, add 250 μL MilliQ water and vortex
for 30 s. Recover the organic phases in a 1.5 mL vial, reextract
the aqueous phase twice with 250 μL of dichloromethane.
14. Dry the pooled organic phases under a nitrogen stream and
store at À20
C until HPLC-PAD analysis (see Note 6).
15. Analyze the presence of carotenoids in the nematode pellet
extracts by using a HPLC-PAD equipped with a C30 analytical
column (4.6 Â 250 mm, 5 μm) couple to a C30 guard column
(20 Â 4.0 mm, 5 μm). In this procedure it is describe the
chromatographic conditions and equipment that is routinely
used in our laboratory which can resolve most of the common
carotenoids present in fruits or fruit-based food. However,
these conditions can be modified and adapted depending on
the carotenoids composition in the extracts under study.
16. Dissolve the nematode pellet extract in 30 μL of methanol–
acetone–chloroform (2:1:3, v/v/v) and centrifuge at
12,300 Â g, 1 min at 20
C.
17. Inject 20 μL on the HPLC system using the method described
elsewhere [33] with a ternary gradient elution. The initial
solvent composition consisted of 90% methanol (MeOH), 5%
water and 5% methyl tert-butyl ether (MTBE). The solvent
composition changes in a linear fashion to 95% MeOH and 5%
MTBE at 12 min. During the next 8 min the solvent composition changes to 86% MeOH and 14% MTBE. After reaching
this concentration the solvent is gradually changed to 75%
MeOH and 25% MTBE at 30 min. Following 20 min solvent
composition changes linearly being, at 50 min, 50% MeOH
and 50% MTBE. The final composition is reached at 70 min
and consisted of 25% MeOH and 75% MTBE. The initial
conditions are established in 5 min and equilibrated for
15 min before the next injection. The flow rate is 1 mL/min,
and the column temperature is set to 25
C. The PAD is set to
scan from 250 to 540 nm, and for each elution a Maxplot
chromatogram is obtained, which plots each carotenoid peak
at its corresponding maximum absorbance wavelength.
18. Compare chromatograms obtained with the pellet extracts of
nematodes grown on NGM-control and NGM supplemented
with carotenoid, and identify carotenoids analyzing the spectra
of the chromatographic peaks (see Notes 16 and 17).
Figure 2b shows chromatograms obtained from C. elegans
grown in NGM control and NGM supplemented with
β-cryptoxanthin at 1 μg/mL. Only one carotenoid peak
which spectrum and retention time matched with
Carotenoids and Oxidative Stress Assay in C. elegans
397