Protocol - JC-1 Assay on sea urchin embryos
Identification of mithocondrial membrane potential under pro- and anti-oxidative stress in sea urchin embryos
Category:
Cell Biology
Creation date: Jan 22, 2010
Last revision: Dec 19, 2014
Author(s):
Marta Di Carlo
Contact
Name: Marta Di Carlo
Address: IBIM CNR via Ugo La Malfa, 153, Palermo
Phone: +39 0916809538
Email: di-carlo@ibim.cnr.it
Figure legend:
P. lividus embryos untreated (red) or treated (green) with an oxidant.
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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JC-1 Assay
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|||
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• 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 (carbonyl cyanide 3-chlorophenylhydrazone) 2μl (50 μM f. c.) to an untreated sample
|
R.T.
|
for 10 minutes
|
|
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• Add JC1 (2μM f. c.) to each sample
|
37
|
for 30 minutes
|
incubation in the dark
|
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• Centrifuge embryos (600 rpm) and eliminate supernatant
|
R.T.
|
10 minutes
|
|
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• Resuspend all the samples 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
|
|
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• Read at the fluorimeter excitation Wavelength (488 nm)
|
R.T.
|
15 minutes
|
emission filters to ~529 nm (green) and ~590 nm (red)
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Other informations
P. lividus embryos should be developped in seawater in Petri dishes (1000 embryos/ml) until to reach the wished embryonic stage
Positive Control: sample treated with CCCP
Negative Control: untreated sample
Quality validation:
Yes
Validation info
The protocol has been published in a peer-reviewed journal.
Solutions
-
JC-1
Reagents F-concentration Quantity I-concentration JC-12 uM -
Sea water millipore filtered
Reagents F-concentration Quantity I-concentration Sea water millipore filtered1 L -
CCCP
Reagents F-concentration Quantity I-concentration CCCP50 uM2 ul -
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
