If I resuspend dihydrorhodamine 123 in EtOH, is it best to store aliquots in -20 or in liquid N2? Also I've seen one recommendation not to use a reconstituted aliquot beyond 1 month. What's the consensus?
Thanks in advance-
Dear Sir. Concerning your issue about what is the best condition to store aliquots of dihydrorhodamine 123. The best conditions are to store at -20°C and to Store under Desiccating conditions. The product can be stored for up to 12 months, and must be in tightly sealed vials . I think the following below attached file may help you in your analysis:
Thanks
Dear Michelle
For our ROS determinations we dissolve the Dihydrorhodamine 123 in DMSO at 2.5 mg/ml and aliquot this as 25 ul volumes into 0.6 ml capped PCR tubes which we briefly gas with N2, seal and store at -80°C.
When required we thaw the number of aliquots required for the assays (flow cytometry or fluorescence spectrometry) and dilute the aliquot to 2.5 ug/ml (total volume 25 ml) in the medium we are to use for the assay. Commonly we use indicator free Hanks balanced salt solution for fluorescence spectrometry and culture media for flow cytometry (as the indicator is diluted away during the assay). This working DHR solution is diluted 1/5 into the actual test samples with the cells or organelles.
Once diluted to 2.5 ug/ml the solution should be kept sealed from the atmosphere on wet ice and is usable for at least the day of dilution. We have kept the diluted DHR solution at -20°C for a day or two which is fine for flow work but the background increases for the fluorescence plate reader work due to the slow oxidation of the DHR. You need to include controls in your assays to compensate for this.
The initial storage at -80°C has never been a problem and storage at lower temperatures would ensure better stability. Storage of the diluted stock should be kept as short as possible so only dilute what you will need plus a bit so that you use the same batch on a particular day. Use a positive and negative control for each experiment to know what the range of the fluorescence would be for that day.
Good luck
Thanks Duncan, that's very helpful. I notice you are using a concentration approximately 10-fold lower than what I am using. (I've only done this twice, and using flow cytometry.) I find that my no-DHR control is truly negative, but that the ice control is shifted above that and then the samples that get a treatment at 37C are all very positive- no way to differentiate between anything meaningful. I'm wondering if I should back my working concentration down.
Hi Michelle
What you are observing is a high conversion rate from the dihydro form of the Rhodamine 123 to the fluorescent oxidized form.
It could be due to the media you are using or for the cellular activity. There is also a maximum signal that the flow cytometer can process so you need to adjust the voltage settings on the instrument to accommodate your negative and a positive control (you can add an equivalent amount of hydrogen peroxide or sodium hypochlorite to a permeabilised cell population) to get the highest signal expected. Check the scale of the fluorescent channel as well as it may be in log scale and then everything looks squashed up but intensity can be differentiated quite well in linear scale.
The signal can then be appropriately gated according to the maximum expected signal and not as an increase from the background. If the signal is still too high then you can reduce the DHR concentration that means new experiments with your cells. There is a point where the concentration of DHR is low enough to be totally oxidized by the background oxidative potential of your cells so you cannot go too low. The concentration does affect the sensitivity of the method.
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