Protocol - ROS assay on sea urchin embryos
Identification of free radical generation under pro- and anti- oxidative stress
Category:
Cell Biology
Creation date: Jan 22, 2010
Last revision: Dec 16, 2014
Author(s):
Picone Pasquale
Contact
Name: Picone Pasquale
Address: IBIM-CNR, via Ugo La Malfa, 153, Palermo
Phone: 0916809546
Email: picone@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.
Solutions
-
Sea water millipore filtered
Reagents F-concentration Quantity I-concentration Sea water millipore filtered900ml -
H2O2
Reagents F-concentration Quantity I-concentration H2O24ul/sample -
DCFH-DA
Reagents F-concentration Quantity I-concentration DCFH-DA1uM1ul1mM -
PBS 1X (pH 7.4)
Reagents F-concentration Quantity I-concentration KCl2.7 mM10 ml/L27 mMNaCl137 mM10 ml/L1.37 MNa2HPO48 mM10 ml/L80 mMKH2PO41.9 mM10 ml/L19 mM
