I have recently added NAO (100-200 nM) to living kidney cells (30 min, 37C) then washed with PBS and immediately visualized with microscopy. I see bright green fluorescence showing typical mitochondrial staining, but it fades/photobleaches very fast. Is this normal? I have not seen this rapid loss of signal stated in publications using NAO. Based on prior publications, I also assume that fluorescence intensity partially correlates with levels of cardiolipin (high fluorescence=high cardiolipin)? Is that a correct assumption? Thank you!!
Mrs/Miss MacMillan-Crow,
Fs spectroscopy does not appear method of choice for such purposes due to limitations of its method performances as analytical tool.
The mass spectrometry appears method of choice providing directly qualitative and quantitative information about the analytes in vivo. In this context, please consider the reference section provided to the followig discussions.
Therein, we have shown our more recently developed quantitative relationships yielding to exact MS determination of physico-chemical and molecular parameters of the analytes, including their concentration in solution.
Discussions:
[1] https://www.researchgate.net/post/How_can_you_determine_if_a_fluorophore_is_adsorbed_to_a_bacterial_cell_or_localized_in_the_cytoplasm
[2] https://www.researchgate.net/post/I_need_to_buy_as_respirometer_for_measuring_the_metabolic_rate_of_insects_I_want_to_know_to_whom_I_have_to_contact
If you have any further questions or requests or both of these associated with the theoretical backround or the application of our method to quantify analytes in complex mixtures, please, do not hesitate to continue the discussion, herein. We shall provide you adequate response, accordingly.
Dear Lee Ann,
prior to answer to your question, I need the following infos:
- What a kind of NAO (supplier, counter anion e.g. bromide or chloride..)
- In what do you solve the NAO salt prior to give it to the kidney cells.
-I send you a link about vital staining of kidney cells ( Year 1928 !!)
https://royalsocietypublishing.org/doi/10.1098/rspb.1928.0042#.XuUTydi1UHE.email
- -give us the protocol related to the washing with PBS (e.g. how many times ..)
-according to your sentence :
but it fades/photobleaches very fast. Is this normal? I have not seen this rapid loss of signal stated in publications using NAO. Based on prior publications, I also assume that fluorescence intensity ...
Please note that the most basic dyes like AO salts and NAO salt are toxic to the most cells and fade e.g. photobleache after excitation . Please read the fading references in Google or Google Scholar.
Good luck
JRG
please read and download the references cited in this link:
Article Tau-induced mitochondrial membrane perturbation is dependent...
and :
https://pubmed.ncbi.nlm.nih.gov/?term=nonyl%20acridine%20orange&page=4&pos=3
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