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Protocol - ROS assay on sea urchin embryos

Identification of free radical generation under pro- and anti- oxidative stress

CategoryCell Biology
Creation dateJan 22, 2010
Last revisionDec 16, 2014
Author(s)Picone Pasquale
Contact
NamePicone Pasquale
AddressIBIM-CNR, via Ugo La Malfa, 153, Palermo
Phone0916809546
Emailpicone@ibim.cnr.it

 

Figure legend:

P. lividus embryo treated with an oxidant molecule


Steps

Description Temperature Time Note
ROS assay
• Fertilize Paracentrotus lividus eggs in seawater containing PABA 2mM
R.T.
10 minutes
• At two cell stage administer pro or anti-oxidants molecules at different concentrations on the basis of literature or preliminary studies
16°C
from 1 hour to 48 hours
• Develop P. lividus embryos until the wished embryonic stage
• Transfer 200μl of untreated and treated embryos in an eppendorf tube
R.T.
10 minutes
• Add CCCP 2μl (50 μM f. c.) to an untreated sample
R.T.
15 minutes
• Centrifuge samples (600 rpm) and eliminate supernatant
R.T.
10 minutes
• Resuspend all the samples in 200μl of PBS 1X
R.T.
10 minutes
• Add 1μl of DCFH-DA (1μM f. c.) at all the samples
R.T.
10 minutes
incubation in the dark
• Centrifuge samples (600 rpm) and eliminate supernatant
R.T.
10 minutes
• Resuspend embryos in 200μl of PBS 1X
R.T.
3 minutes
• Split all the samples in two wells (double) of a 96 wells black plate
R.T.
15 minutes
• Read at the fluorimeter excitation Wavelength (488 nm)
R.T.
15 minutes
emission filters to ~529 nm (green)

Other informations

P. lividus embryos should be allowed to develop in sea water in Petri dishes  (1000 embryos/ml) until to reach the wished embryonic stage.

Positive Control: sample treated with H2O2

Negative Control: untreated sample

 

Quality validation: Yes

Validation info

The protocol has been published in a  peer-reviewed journal.

 

Citations
Picone P, Nuzzo D, Di Carlo M. Ferulic acid: a natural antioxidant against oxidative stress induced by oligomeric A-beta on sea urchin embryo. Biol. Bul. 2013;224:18-28.

 

Solutions

  • Sea water millipore filtered

    Reagents F-concentration Quantity I-concentration
    Sea water millipore filtered
    900ml

  • H2O2

    Reagents F-concentration Quantity I-concentration
    H2O2
    4ul/sample

  • DCFH-DA

    Reagents F-concentration Quantity I-concentration
    DCFH-DA
    1uM
    1ul
    1mM

  • PBS 1X (pH 7.4)

    Reagents F-concentration Quantity I-concentration
    KCl
    2.7 mM
    10 ml/L
    27 mM
    NaCl
    137 mM
    10 ml/L
    1.37 M
    Na2HPO4
    8 mM
    10 ml/L
    80 mM
    KH2PO4
    1.9 mM
    10 ml/L
    19 mM

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