I have done previously ROS by DHE stain using flowcytometry. My study design included adherent cell culture: Cells after seeding, treatment, trypsinizing, pellet was incubated with FBS-free medium + DHE dye for 15 min in dark at room temperature. Then centrifuged and washed with chilled PBS, and read using PBS in flowcytometer.
Now i want to try using fluorescent microplate reader.. any reliable protocol that I can follow?
What kind of buffer i should use to read results?
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For adherent cell culture you can do almost the same, just instead labeling with DHE after trypsinizing you need to incubate adhered cell with DHE, then you can wash that medium, add trypsine and without taking that suspension you add ice cold PBS to each well, you can read immeadiately, this will be a homogeneus suspension with the same amount of cells you have when labeling end.
Hope this works for you. Good luck
Thanks alot Paola..
I have two inquiries here, if possible:
1) Is it necessary to have a homogeneous suspension of cells? Cuz I read some protocols taking readings of cells still attached to plate, after incubation with dye.
2) if I add trypsin to cells in wells, then just add PBS.. the continuous un-stopped trypsin action wont affect cells? Usually we add media to neutralize trypsin, and prevent harm to cells.
Appreciate your feedback :)
Hello, did you try Amplex Red? You can use it in adherent cell cultures..
1) Yes, you can read adhered cells, however if you have less confluence in some wells you need to make sure microplate reader do analyze the entire area, otherwise measure may be lower not by less ROS but by less homogeneous distribution along the well... what I saw was a more consistent result with suspension. Just add a shaking step before to read.
2) Yes, if you add just PBS trypsin will be still active and it can lyse proteins from your cells, however if you add ice cold PBS trypsine won't have the same activity and cells won't be damaged. I didn't add supplemented medium because that is a complex mixture that can interfere with your readings.
3) You can try some other reagents, I did it with dichlorofluorescein and worked for me. Didn't check with DHE or Amplex Red, but if you already have information from flow cytometry probably you can have good results with it on microplate reader as well.
Good luck! :-)
Thanks Clarissa, but since I have DHE in lab, I prefer to try it first. Good information though , thanks!
Thank you very much Paola, I understand now the steps you have done, and will try to apply and see how it goes.
Did you publish any paper using this method for ROS detection? if so pls share the link herein, or share any paper that can be used as a reference for this method, if possible to do that.
Really appreciate your advice, thanks again :)
Paola Ibelles, how do you normalize the fluorescence? Do you check the protein concentration or just make sure that you plate the same number of cells per well?
This would be a problem for me as my knock down model grows much faster than my control cell. I tried normalize with Hoechst but DHE intercalates with DNA and increases the Hoechst sign, leading to an altered and strange results.
I am sure Paola is more expert in this, I would also like to know her opinion.
For me, previously I included a cell count step just before adding the dye for control and tested samples (after trypsinizing cells) and standardize cell count in all wells, then added the dye.
Hope it helps a bit.
I did it to compare conditions which didn't affect cell viability, so just equally seeding cells from same cell suspension was enough to have same cell number and didn't need to further standarize. Also, I did an exact duplicate for plates and measure viability with lactate dehydrogenase realease, if you do have different viable cell number I guess you can use that to standarize.
Lina: My paper is still having the fight out there to be published so the best I can share is one of the papers I read, I did some adjustments, but those I already explained to you.
J Immunol Methods. 1992 Nov 25;156(1):39-45.
A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate.
Rosenkranz AR1, Schmaldienst S, Stuhlmeier KM, Chen W, Knapp W, Zlabinger GJ.