Hi,
I have been trying to set the CellROX measure in hematopoietic stem cells, but without further success.
I stained my cells before the CellROX incubation. And I used the controls that came with the kit NAC (500 nM) and THBP (400 nM). Then, I've tried the the following concentrations of CellROX 500 nM and 1 uM for 30 minutes at 37 degrees celsius (water bath) in medium. At the end of the treatments I fix my cells, and I measure the CellROX by cytometry.
The results have been inconsistent. The staining doesn't last, and the CellROX does not produce higher signal in presence of THBP.
If someone has used this, please could you give me some advice/feedback?
Thank you so much,
Have a nice day!
HI,
You can not fix the cells. They should be run on the flow cytometer as possible after the 30min incubation at 37C. Place them on ice if there is more than a 10 min delay between incubation and analysis, but do not wait more than one hour.
Kind regards,
Steve
HI Sarah,
You can try another approach for measuring intracellular ROX. DCF is for total ROX, while Mitosox is for intramitochondrial ROX. These methods are really easy and fast. You can find details in one of my article:
Molecular pharmacology 07/2014; DOI: 10.1124/mol.114.094318
Kind regards,
Attila
Hi Attila,
I have been using DCFDA as it measures hydrogen peroxyde in the cell.
I have been also recently using the Mitosox for the superoxyde anions specific to mitochondria.
But I thought about CellROX as I was trying to find a more sensitive assay, I got some feedback of people about the cellrox, as it measures total superoxyde anions.
The mitosox it goes fine, but I don't find it very sensitive as in my hands, as I need a massive build up of ROS to be able to see a signal. When I incubate the cells with higher doses of oxidant, I can see higher mitosox signal. I work with hematopoietic stem cells, and the active mitochondria content is very low...
Anyway I proceed in a similar way for mitosox incubating the cells in HBSS for 15 minutes in water bath at 37 degrees celsius.
Do you have some advice about increasing the sensitivity of the mitosox?
Thank you Steve, I have tried without fixing. My first assays with mature cells or progenitors were good, with fixation. As when I put on ice the control sample tend to increase their ROS. And with the fixation the signal was more stable.
Sorry to tag onto this. But I am about to start using CellRox and wondered if anyone can explain the need for the 30min 37 degree step. Can this be adapted to improve signal? and is 37 essential, my cell line is maintained at 26 degree so i worry this might stress the cells and result in ROS background
From my experience with MitoSox (I believe it applies to CellRox as well) I assume that 37C step is just to incubate cells in most "comfortable" for the cells conditions to get permeable cell dye in. I also always use viability dye (for ex. DAPI with MitoSox Red staining) for flow cytometry as I noticed that dead and apoptotic cells are highly MitoSox positive. This may explain why Reactive Oxygen Species dyes do not work well for hematopoietic stem cells. Also HSCs have active pumps that push all kind of dyes out of the cells.
For cells normally grown at 26 degrees I would likely incubate with dye at 26C. You are absolutely right about being concerned about 37C conditions. If cells cannot be analyzed immediately and fixation step is needed I would recommend to stain cells first with fixable viability dyes (like Aqua Dead, Zombie Nir, etc depending on particular MitoSox / CellRox ex/em spectrum)) to discriminate between "live" and "dead" cells. It works well for flow cytometry and is more challenging for microscopy due to wide emission/excitation spectrum of ROS dyes and hence spillover into neighboring channels and special filters requirements.
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