I remember we had been using Acridine orange as a nucleic acid dye in malaria studies. I cannot remember when and especially why we switched to using EtBr as a nucleic acid dye in malaria studies.
EtBr is know to be a dangerous dye and poses a health risk to humans. Is there an alternative for it's use in malaria studies.
Regretfully, I do not know the answer, because I do not study Plasmodium infected RBC. However, I will gladly ask a colleague at the institute and respond later.
It will depend a bit on what experiment you are running. Do you need the parasites to be live or will they be dead at the time of analysis. SYBRGreen is an excellent nucleic acid stain, though mostly used for flow cytometry, and of course if you are double labelling, be sure to avoid anything else using the 488mn wavelength. If you have the right lazer, DAPI (4',6-diamidino-2-phenylindole) is also an excellent stain, I have often used it for live cell imaging.
How are you studying the dyed cells? Microscopy or Flow cytometry? Are you wanting to enumerate parasites or visualise them?
Its better use Acridine Orange, if you are using flow cytometry to quantify parasitemia in detail because this is pretty much the only dye that can distinguish between trophs and rings. However, there are several other cell permeable DNA dyes you can use instead of EtBr if you are just interested in obtaining parasitemia numbers. You can use DAPI or Hoechst or any of the SYTO dyes. The SYTO dyes come in many flavors with different emission/excitation spectra. You should also know that EtBr is perfectly safe if you handle it with proper protective gear like always using gloves.
Sorry for not indicating that these studies are on Flow cytometer. One may use Cyan ADP for acquisition of DAPI stained cells because of the presence of purple laser. Due our internal policy not to use infected red Cells on Cyan. I will have to test the rest of the dyes you have suggested for me to use. Again thanks alot for your in put.
Hi..
I have used DAPI and it works fine. Probably, you can give it a try.
You should consider using the method outlined in the following paper. I use this method, as the author is within my institute and has shown me personally how to use it. It works great with the Hoechst dye, and you can further differentiate RBCs and reticulocytes using anti-TER119 and CD71 (in mouse). I assume you are using this for P. falciparum cultures though? I know of some who have adapted this method using Pf.
http://www.sciencedirect.com/science/article/pii/S0020751911002177
DAPI, Hoechst stains, Acridine orange (AO) and benzothiocarboxypurine (BCP) are all good nuclear stains.Both AO and BCP are excitable @ 490nm and exhibit apple green or yellow fluorescence.
Rhodamine-123 is a dye which labels only viable parasite membrane and its accumulation into the inside of the parasite is dependent on the membrane potential. The potential can only be maintained in viable parasite with functional electrogenic H+ ATPase.
Hi Oscar, As fixation/permeabilisation can have numerous effects on the evaluation of malaria levels - eg differential lysis, aggregation - so we try to use permeant dyes for the evaluation of parasitemia. Staining is done in derogated labs, at the end of the appropriate dye incubation period, diluent containing sodium azide or paraformaldehyde is added, to mitigate hazard potential, before transport to the flow facility for immediate acquisition. Hoechst is an excellent stain for malaria ,where UV excitation is available, but does not give sufficient resolution, when excited by a standard violet laser, to clearly distinguish infected cells from uninfected rbc. DAPI staining is poor for rings, even with UV excitation, cf. Hoechst. Hydroethidine gives similar results to EtBr, at much lower concentrations, due to it's higher permeability but requires the parasite to be metabolically active to oxidise the dye to stain DNA. Thiazole orange predominantly stains RNA, and like DAPI is poor for staining early stages of infection. DRAQ dyes appear to work but have not been heavily evaluated due to their relative cost.
Finally acridine orange is not usually permitted by flow managers, as it will stain the instruments fluidic tubing and interfere with subsequent acquisitions EVEN AFTER flushing with bleach!
Acridine Orange stains the nucleus of the parasite, which can replace eb. lalitha kabilan
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