β-cryptoxanthin, was identified in the nematodes grown in
NGM supplemented with β-cryptoxanthin, confirming the
carotenoid uptake by the animal.
4 Notes
1. To avoid variability between trials it is important to be sure that
the same batch of test compounds will be used along the
experiment. This is particularly determinant when a carotenoid
extract is evaluated since may contain a complex and not
defined proportion of carotenoids.
2. When possible used the highest purity standard available
(>95%) to avoid impurities with other carotenoids.
3. It is possible to assay a novel carotenoid that has been purified
by HPLC or TLC.
4. It is important to completely dissolve C or CE. To that end,
follow the solvents recommended elsewhere [34] since this can
be variable depending of the polarity of your carotenoid, and
determine concentration spectrophotometrically as described
elsewhere [34]. Be sure that solvents used do not contain
antioxidants such as butylated hydroxytoluene which will interfere in the assay.
5. We recommend using a Hamilton syringe to take the aliquot
since it is important to accurately measure the volume.
6. Do not store dried carotenoids or extracts for more than
2 weeks. In our experience if sample to evaluate contains lycopene or phytoene do not store for more than 2 days since these
carotenes are more unstable.
7. The range of concentrations must be adjusted to each carotenoid/carotenoid extract, and these concentrations corresponds
assays performed to β-cryptoxanthin, lycopene and β-carotene
in our laboratory. However, we recommend to test in exploratory experiments doses up to 2 μg/mL for pure carotenoids
and up to 10 μg/mL for complex extracts.
8. The solvent used to dissolve the C or CE (e.g., chloroform–
ethanol) can cause deleterious effects on C. elegans performance, therefore the same amount of solvent should be
added to control NGM dishes.
9. Nematodes are transferred to new plates using a platinum wire.
10. It is possible to further explore the cellular and molecular
mechanism involved in the C. elegans response to carotenoid
treatment and subsequent oxidative stress by means of transcriptional profile or biochemical (e.g., ROS production)
changes in the nematodes. To that end the number of dishes
398
Patricia Martorell et al.
NGM supplemented with β-cryptoxanthin, confirming the
carotenoid uptake by the animal.
4 Notes
1. To avoid variability between trials it is important to be sure that
the same batch of test compounds will be used along the
experiment. This is particularly determinant when a carotenoid
extract is evaluated since may contain a complex and not
defined proportion of carotenoids.
2. When possible used the highest purity standard available
(>95%) to avoid impurities with other carotenoids.
3. It is possible to assay a novel carotenoid that has been purified
by HPLC or TLC.
4. It is important to completely dissolve C or CE. To that end,
follow the solvents recommended elsewhere [34] since this can
be variable depending of the polarity of your carotenoid, and
determine concentration spectrophotometrically as described
elsewhere [34]. Be sure that solvents used do not contain
antioxidants such as butylated hydroxytoluene which will interfere in the assay.
5. We recommend using a Hamilton syringe to take the aliquot
since it is important to accurately measure the volume.
6. Do not store dried carotenoids or extracts for more than
2 weeks. In our experience if sample to evaluate contains lycopene or phytoene do not store for more than 2 days since these
carotenes are more unstable.
7. The range of concentrations must be adjusted to each carotenoid/carotenoid extract, and these concentrations corresponds
assays performed to β-cryptoxanthin, lycopene and β-carotene
in our laboratory. However, we recommend to test in exploratory experiments doses up to 2 μg/mL for pure carotenoids
and up to 10 μg/mL for complex extracts.
8. The solvent used to dissolve the C or CE (e.g., chloroform–
ethanol) can cause deleterious effects on C. elegans performance, therefore the same amount of solvent should be
added to control NGM dishes.
9. Nematodes are transferred to new plates using a platinum wire.
10. It is possible to further explore the cellular and molecular
mechanism involved in the C. elegans response to carotenoid
treatment and subsequent oxidative stress by means of transcriptional profile or biochemical (e.g., ROS production)
changes in the nematodes. To that end the number of dishes
398
Patricia Martorell et al.