Methodology Flashcards
What range of ROS concentrations can DCF detect?
DCF fluorescence can change measurably at 10 pM concentrations for some detection kits by Cell BioLabs.
What are some dyes that can be used to measure ROS?
Dichlorodihydrofluorescein, hydroethidine, and dihydrorhodamine (Kaylanaraman, 2012).
In simple terms, what is the transition of DCFH to its fluorescent state?
DCFH undergoes two-electron oxidation to become the fluorescent DCF (Kaylanaraman, 2012).
Can DCF generation be used to measure hydrogen peroxide?
No DCFH is oxidized by one-electron-oxidizing species to DCF- and then by oxygen to DCF. In fact DCF- to DCF seems to generate H2O2 (Kalyanaraman, 2012).
(Kaylanaraman, 2012)
A review on fluorescence techniques for measuring ROS. Primarily looks at DCF-DA.
What are the six limitations of DCFH?
1) Does not react directly with Hydrogen Peroxide.
2) DCFH is oxidized by hydroxyl radicals, peroxidase or heme’s interacting with H2O2, NO2 radical from myeloperoxidase/H2O2/NO2- system, and reactive species from peroxynitrite decomposition. Peroxynitrite decomposition also forms hydroxyl and carbonate anion radicals when bicarbonate is present.
3) Intermediate DCF radical rapidly reacts with O2 (10^8/M/s) to form superoxide, which dismutates to yield additional H2O2. Can lead to redox-cycling and artifactual amplification of fluorescence signal intensity.
4) Cyt. c is a heme protein released from mitochondria during apoptosis. It also directly and indirectly through peroxidase, oxidizes DCFH to DCF. Therefore it may make it look like ROS levels increase during apoptosis.
5) Redoxi-active metals promote DCFH oxidation in the presence of oxygen or H2O2.
6) Control and experimental samples may not have the same efficiency of radical generation or self-propagating redox-cycling reactions induced by the DCF radical.
(Kaylanaraman, 2012)
Why is it problematic to interpret an increase in DCF fluorescence during apoptosis?
Cyt. c gets released from mitochondrion during apoptosis, which itself oxidizes DCFH to DCF (Kaylanaraman, 2012).
What is DCF useful for?
It’s good as a general indicator of redox state or changes in intracellular iron signaling or peroxynitrite formation (Kaylanaraman, 2012).
May be good for detecting Cyt. c release from mitochondria as well.
What effect does rhodamine-123 have on mitochondrion?
At high concentrations it inhibits mitochondrion electron transport chain by binding membranes. It can also inhibit transport processes.
At what concentration does rhodamine-123 fluorescence peak? Why does fluorescence decrease after this point?
Rhodamine-123 fluorescence peaks at 50 uM and decreases after due to self-quenching (Huang, 2007).
(Chen, 1982)
First showed that rhodamine-123 stained mitochondria.
What is the relationship between rhodamine-123 fluorescence and mitochondrial membrane potential?
There is a linear relationship between fluorescence and membrane polarization shown qualitatively by Emaus et al.
However Huang’s model from 2007 predicts a sigmoid relationship between fluorescence intensity and dye concentrations below 19 uM. Beyond this relationship isn’t monotonic anymore due to quenching.
What is the relative concentration ratio between rhodamine-123 inside mitochondria versus rhodamine-123 concentration in cytosol?
Based on the Nernst equation, between 342 and 2400 (Huang, 2007).
What can potentially be used as a positive control rhodamine-123 measurements?
Can depolarize mitochondria to 0 mV using CCCP (collapse proton gradient).
How much rhodamine-123 stays bound to mitochondria instead of moving back and forth between the matrix and cytosol?
60% total rhodamine-123 is expected to be membrane bound.
