specific procedure to assess the carotenoid/s intake by the nematode by using an HPLC-PAD system. This protocol can be particularly useful for screening a high number of samples and to
compare the protection capacity between them. Furthermore, the
procedures can easily be adapted to evaluate the effect of carotenoid
supplementation or enrichment in complex food matrices.
2 Materials
Use analytical or, when indicated, HPLC grade reagents, and ultrapure water (MilliQ, prepared by purifying deionized water). Work
in the fume cupboard when using organic solvents. For manipulation of bacteria and nematodes work with sterile material and
conditions (e.g., laminar flow cabinet). Carotenoids solutions and
extracts should be maintained under dim light conditions, and
store solutions and dried extracts under nitrogen atmosphere to
prevent carotenoid oxidation.
2.1 Caenorhabditis
elegans Cultivation
and Manipulation
1. C. elegans strain N2, Bristol (wild-type) is provided by the
Caenorhabditis Genetics Center (University of Minnesota,
Minneapolis, MN, USA). C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar(NaCl,
agar, peptone, 5 mg/mL cholesterol, 1 M KPO 4 buffer
pH 6.0, 1 M MgSO 4 ), aseptically poured into petri dishes, at
20
C. Plates are seeded with an Escherichia coli OP50 (University of Minnesota, C. elegans Genetics Center) bacteria lawn
as food source.
2. Transferring nematodes grown on NGM plates is performed
using a dissecting stereomicroscope, picking single animals
with a worm picker (a platinum wire) which is flamed between
transfers to avoid contaminations.
2.2 Reagents,
Solutions, and Buffers
1. NGM solution and preparation of NGM petri dishes
(60 Â 15 mm) with E. coli OP50. For 1 L of NGM solution,
add 0.75 g Bacto peptone, 0.9 g NaCl, 5.1 g Bacto agar,
0.3 mL cholesterol solution (prepared by dissolving 1 g of
cholesterol in 200 mL of 100% ethanol, not autoclaved and
stored at room temperature). Add distilled water to 292 mL
and autoclave at 120
C, 20 min. After autoclaving, add 0.3 mL
of 1 M CaCl 2 , 0.3 mL of 1 M MgSO 4 and 7.5 mL of 1 M Kphosphate (pH 6). Keep it at 60
C until preparing the dishes.
Pour 10 mL of NGM solution into each petri dish and leave at
room temperature from several hours to overnight. Make an
overnight culture of OP50 with LB and add 50 μL of the OP50
solution to each NGM dish and leave overnight at room
temperature.
Carotenoids and Oxidative Stress Assay in C. elegans
391
compare the protection capacity between them. Furthermore, the
procedures can easily be adapted to evaluate the effect of carotenoid
supplementation or enrichment in complex food matrices.
2 Materials
Use analytical or, when indicated, HPLC grade reagents, and ultrapure water (MilliQ, prepared by purifying deionized water). Work
in the fume cupboard when using organic solvents. For manipulation of bacteria and nematodes work with sterile material and
conditions (e.g., laminar flow cabinet). Carotenoids solutions and
extracts should be maintained under dim light conditions, and
store solutions and dried extracts under nitrogen atmosphere to
prevent carotenoid oxidation.
2.1 Caenorhabditis
elegans Cultivation
and Manipulation
1. C. elegans strain N2, Bristol (wild-type) is provided by the
Caenorhabditis Genetics Center (University of Minnesota,
Minneapolis, MN, USA). C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar(NaCl,
agar, peptone, 5 mg/mL cholesterol, 1 M KPO 4 buffer
pH 6.0, 1 M MgSO 4 ), aseptically poured into petri dishes, at
20
C. Plates are seeded with an Escherichia coli OP50 (University of Minnesota, C. elegans Genetics Center) bacteria lawn
as food source.
2. Transferring nematodes grown on NGM plates is performed
using a dissecting stereomicroscope, picking single animals
with a worm picker (a platinum wire) which is flamed between
transfers to avoid contaminations.
2.2 Reagents,
Solutions, and Buffers
1. NGM solution and preparation of NGM petri dishes
(60 Â 15 mm) with E. coli OP50. For 1 L of NGM solution,
add 0.75 g Bacto peptone, 0.9 g NaCl, 5.1 g Bacto agar,
0.3 mL cholesterol solution (prepared by dissolving 1 g of
cholesterol in 200 mL of 100% ethanol, not autoclaved and
stored at room temperature). Add distilled water to 292 mL
and autoclave at 120
C, 20 min. After autoclaving, add 0.3 mL
of 1 M CaCl 2 , 0.3 mL of 1 M MgSO 4 and 7.5 mL of 1 M Kphosphate (pH 6). Keep it at 60
C until preparing the dishes.
Pour 10 mL of NGM solution into each petri dish and leave at
room temperature from several hours to overnight. Make an
overnight culture of OP50 with LB and add 50 μL of the OP50
solution to each NGM dish and leave overnight at room
temperature.
Carotenoids and Oxidative Stress Assay in C. elegans
391