2. M9 buffer is used to recover, washing and transfer high number
of worms, eggs or larvae. For 1 L of buffer, add 3 g KH 2 PO 4 ,
6 g Na 2 HPO 4 , 5 g NaCl, 1 mL 1 M MgSO 4 . Sterilize by
autoclaving.
3. Basal S medium is used for the oxidative stress. For 1 L, add
5.85 g NaCl, 17 g agar. After autoclave, add 50 mL of 1 M Kphosphate buffer (pH 6) and 1 mL cholesterol (prepared by
dissolving 1 g of cholesterol in 200 mL of 100% ethanol, not
autoclaved and stored at room temperature).
4. Liquid nitrogen.
5. Hydrogen peroxide 30% stock for acute oxidative stress. Store
stock at 4
C.
6. Nitrogen gas.
7. Micropipettes, Hamilton syringes, 1.5 mL Eppendorf type
vials, 2 mL cryovials, 50 mL tubes, QS 1 mL cuvettes and
platinum wire (for nematode manipulation).
2.3 Equipment
1. Stereomicroscope equipped with a transmitted light source
(e.g., Motic SMZ168) to score animal features like size or
motility. View the nematodes directly on plates on the stage
of the stereomicroscope.
2. UV-Vis spectrophotometer with cuvette port (e.g., SPECTROstar Nano, BMG Labtech) to determine the concentration of the carotenoid in a solution or extract.
3. Growth chamber at 20
C.
4. Laminar flow cabinet.
5. Centrifuge (for 1.5 mL vials and 50 mL tubes) and vortex.
6. Gas extraction cabinet to work with organic solvents.
3 Methods
3.1 Preparation
of Pure Carotenoid
Solution (C) or
Carotenoids Extract
(CE) to Assay
The first step in this procedure is to obtain a dried aliquot of C or
CE that will be dissolve in a suitable solvent and volume immediately before is added to the NGM dishes. The amount of C or CE
necessary to perform a representative oxidative stress assay can
range between 10 and 50 μg (see Note 1). The source of the C or
CE to be tested can be commercial (see Note 2) or previously
purified from any food or biological source (see Note 3).
1. Prepare a stock solution of C or CE using appropriate organic
solvents (e.g., chloroform, dichloromethane, methanol, or acetone) to reach a final concentration between 100 and 300 ng/
mL determined by spectrophotometer (see Note 4).
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Patricia Martorell et al.
of worms, eggs or larvae. For 1 L of buffer, add 3 g KH 2 PO 4 ,
6 g Na 2 HPO 4 , 5 g NaCl, 1 mL 1 M MgSO 4 . Sterilize by
autoclaving.
3. Basal S medium is used for the oxidative stress. For 1 L, add
5.85 g NaCl, 17 g agar. After autoclave, add 50 mL of 1 M Kphosphate buffer (pH 6) and 1 mL cholesterol (prepared by
dissolving 1 g of cholesterol in 200 mL of 100% ethanol, not
autoclaved and stored at room temperature).
4. Liquid nitrogen.
5. Hydrogen peroxide 30% stock for acute oxidative stress. Store
stock at 4
C.
6. Nitrogen gas.
7. Micropipettes, Hamilton syringes, 1.5 mL Eppendorf type
vials, 2 mL cryovials, 50 mL tubes, QS 1 mL cuvettes and
platinum wire (for nematode manipulation).
2.3 Equipment
1. Stereomicroscope equipped with a transmitted light source
(e.g., Motic SMZ168) to score animal features like size or
motility. View the nematodes directly on plates on the stage
of the stereomicroscope.
2. UV-Vis spectrophotometer with cuvette port (e.g., SPECTROstar Nano, BMG Labtech) to determine the concentration of the carotenoid in a solution or extract.
3. Growth chamber at 20
C.
4. Laminar flow cabinet.
5. Centrifuge (for 1.5 mL vials and 50 mL tubes) and vortex.
6. Gas extraction cabinet to work with organic solvents.
3 Methods
3.1 Preparation
of Pure Carotenoid
Solution (C) or
Carotenoids Extract
(CE) to Assay
The first step in this procedure is to obtain a dried aliquot of C or
CE that will be dissolve in a suitable solvent and volume immediately before is added to the NGM dishes. The amount of C or CE
necessary to perform a representative oxidative stress assay can
range between 10 and 50 μg (see Note 1). The source of the C or
CE to be tested can be commercial (see Note 2) or previously
purified from any food or biological source (see Note 3).
1. Prepare a stock solution of C or CE using appropriate organic
solvents (e.g., chloroform, dichloromethane, methanol, or acetone) to reach a final concentration between 100 and 300 ng/
mL determined by spectrophotometer (see Note 4).
392
Patricia Martorell et al.