per condition should be extended to 50 and upon treatment
nematodes are collect from the dishes, follow steps 4–8 from
Subheading 3.4, and immediately frozen in liquid nitrogen and
store at À80
C until analysis.
11. The H 2 O 2 is added to the autoclaved medium ensuring a
temperature below 50
C.
12. If it has been previously tested the effectiveness of the C or CE
in a dose-response experiment, the dishes for the bioassimilation assays can be prepared at the most effective dose. However, in our experience, determination of carotenoids by
HPLC-PAD in nematodes fed with pure carotenoids at doses
below 0.1, are usually below the detection limits. Therefore,
the dose in the dishes can be increase up to 1–5 μg/mL since
the only purpose of this experiment is to confirm the carotenoids uptake and not to evaluate the biological response of
nematodes.
13. This washing is necessary to remove the E. coli OP50 present in
the media that could interfere in subsequent determinations.
14. We recommend to take a picture of the nematode pellets in the
cryovials prior to freezing. Usually the pellet of nematodes
grown in NGM supplemented with carotenoids show a creamy,
yellow or pale orange color compared to the translucent aspect
of NGM-control. See Fig. 2a for an example of a nematode
feeding experiment with β-cryptoxanthin.
15. By weighing the cryovials before and after transferring the
nematodes, the nematode pellet weight can be determined,
and usually is about 100–200 mg.
16. It is recommended to analyze the carotenoid or carotenoid
extract use for nematode feeding by using the same chromatographic conditions. This allows a direct comparison facilitating the identification of carotenoids in the nematode
extracts.
17. If the nematode pellet is weighed before extraction as suggested in step 8 (see Note 15), the relative carotenoid uptake
by the nematode can be determined per mg of nematode pellet
and compared between experiments or conditions.
Acknowledgments
Financial support by research grant AGL-2015-70218 (Ministerio
Economı ´a y Competitividad, Spain) is gratefully acknowledged.
MJR and LZ are members of Eurocaroten (COST_Action
CA15136) and CaRed (Spanish Carotenoid Network, BIO201571703-REDT and BIO2017-90877-REDT).
Carotenoids and Oxidative Stress Assay in C. elegans
399
nematodes are collect from the dishes, follow steps 4–8 from
Subheading 3.4, and immediately frozen in liquid nitrogen and
store at À80
C until analysis.
11. The H 2 O 2 is added to the autoclaved medium ensuring a
temperature below 50
C.
12. If it has been previously tested the effectiveness of the C or CE
in a dose-response experiment, the dishes for the bioassimilation assays can be prepared at the most effective dose. However, in our experience, determination of carotenoids by
HPLC-PAD in nematodes fed with pure carotenoids at doses
below 0.1, are usually below the detection limits. Therefore,
the dose in the dishes can be increase up to 1–5 μg/mL since
the only purpose of this experiment is to confirm the carotenoids uptake and not to evaluate the biological response of
nematodes.
13. This washing is necessary to remove the E. coli OP50 present in
the media that could interfere in subsequent determinations.
14. We recommend to take a picture of the nematode pellets in the
cryovials prior to freezing. Usually the pellet of nematodes
grown in NGM supplemented with carotenoids show a creamy,
yellow or pale orange color compared to the translucent aspect
of NGM-control. See Fig. 2a for an example of a nematode
feeding experiment with β-cryptoxanthin.
15. By weighing the cryovials before and after transferring the
nematodes, the nematode pellet weight can be determined,
and usually is about 100–200 mg.
16. It is recommended to analyze the carotenoid or carotenoid
extract use for nematode feeding by using the same chromatographic conditions. This allows a direct comparison facilitating the identification of carotenoids in the nematode
extracts.
17. If the nematode pellet is weighed before extraction as suggested in step 8 (see Note 15), the relative carotenoid uptake
by the nematode can be determined per mg of nematode pellet
and compared between experiments or conditions.
Acknowledgments
Financial support by research grant AGL-2015-70218 (Ministerio
Economı ´a y Competitividad, Spain) is gratefully acknowledged.
MJR and LZ are members of Eurocaroten (COST_Action
CA15136) and CaRed (Spanish Carotenoid Network, BIO201571703-REDT and BIO2017-90877-REDT).
Carotenoids and Oxidative Stress Assay in C. elegans
399