I wanted to test my cells ROS production by Reactive Oxygen Species (ROS) Detection Reagents (invitrogen), but I did not get positive results even with my control (PMA treated), ROS is lower than unstained cells. The reagent is not the problem because someone used before and got results.
The PMA treated I used is 50uM for 4 hours and while using flow cytometer, the living cells is enough for detection.
I'm surprised you had any cells still alive (actually, they may be dead and this might explain your results). 50uM PMA is a lot, and 4 hours is quite long. With neutrophils and macrophages, 20-100nM PMA is sufficient to generate ROS within 10 seconds, and this is sustained for at least 10 minutes.
I am not sure which reagent you used; CM-H2DCFDA should work fine using FACS. As a positive control, you may try 100uM H2O2 treatment instead of PMA.
Dihydrorhodamine 123 or CellROX reagents should work as well. Depending on what cell type you are using, a 4 H stimulation with PMA may be too long. I've done as short as 15 minutes with primary neutrophils and see substantial signal with Dihydrorhodamine 123.
First, what kind of cells are you testing this in? Second, I would use a positive control that will give you a stronger signal, like UV or H2O2. Third, does PMA refer to TPA? This does not cause ROS production in all cells. Fourth, how long do the cells sit around while you prep for your flow? ROS responses usually occur very rapidly following treatment, so I agree that 4 hours might be to long to wait. Some detection methods allow you fix your cells while others require a live cell assay format. CFDA works well to detect ROS by FACS.
I'm surprised you had any cells still alive (actually, they may be dead and this might explain your results). 50uM PMA is a lot, and 4 hours is quite long. With neutrophils and macrophages, 20-100nM PMA is sufficient to generate ROS within 10 seconds, and this is sustained for at least 10 minutes.
Evan is right!!! High concentrations of PMA might be reducing cellular viability. This might explain why you have lower signals compared to your control cells. I use 100nM of PMA for 10min and it works fine.
Thank you everyone!! I am very grateful for all of your answers.I am sorry for that I haven't express clearly about my issue.Now, please allow me to explain more details.And sadly, in recent two weeks, this problem haven't been solved and I am frustrated entirely.
1. Firstly, I made a mistake that I use the concentrations of PMA is 50nM, not 50 uM.I checked my research data and sure about it.After that, I was wondering the conc. is low so I tried 80nM and 100nM. but all of them can not be positive control. Thanks Fabianno and Evan. Can you help me more?
2. What about the time of PMA-treated? Since others in our lab had tried before, and she used 4 hours and the same cell line to me. It is very strange that why she can figure out positive and I can not. Thanks Ryan and Evan, I am wondering whether I should shorten the time or not.
3. My cell line is RAW264.7---a macrophage cell line. I have used THP-1 cell line before and find the flurorescent is obvious, which can make sure the H2DCFDA is fine.But I can not sure whether the PMA is useful, you know, for the background is very high. Thank you, Carl and Anne. Can you provide me more advises?
4. We used the H2DCFDA(D399. Invitrogen), and the store concentration of it is 200mM, the working concentration of it is 10uM.
This is more details about my issue, and I still cannot figure out now. Can you provide me more advises? It will be grateful for all of you.
Thank you everyone!! I am very grateful for all of your answers.I am sorry for that I haven't express clearly about my issue.Now, please allow me to explain more details.And sadly, in recent two weeks, this problem haven't been solved and I am frustrated entirely.
1. Firstly, I made a mistake that I use the concentrations of PMA is 50nM, not 50 uM.I checked my research data and sure about it.After that, I was wondering the conc. is low so I tried 80nM and 100nM. but all of them can not be positive control. Thanks Fabianno and Evan. Can you help me more?
2. What about the time of PMA-treated? Since others in our lab had tried before, and she used 4 hours and the same cell line to me. It is very strange that why she can figure out positive and I can not. Thanks Ryan and Evan, I am wondering whether I should shorten the time or not.
3. My cell line is RAW264.7---a macrophage cell line. I have used THP-1 cell line before and find the flurorescent is obvious, which can make sure the H2DCFDA is fine.But I can not sure whether the PMA is useful, you know, for the background is very high. Thank you, Carl and Anne. Can you provide me more advises?
4. We used the H2DCFDA(D399. Invitrogen), and the store concentration of it is 200mM, the working concentration of it is 10uM.
This is more details about my issue, and I still cannot figure out now. Can you provide me more advises? It will be grateful for all of you.
I can tell you that I had a big fight with PMA and ROS detection as positive control too. I am using a bit different model, because I use zebrafish and it was hard to find a concentration in which ROS are produced and fish do survive. I found that 100ng/ml of PMA works perfectly. I also use H2DCFDA dissolved in DMSO (final concentration is 5uM). The thing is that I keep my fish... 20 hours in this concentration of PMA, but first changes between negative and positive control can be seen about 4-6 hours (I would need to check my data, because I don't remember now).
I need some help regarding DHR assay. When I am setting up an experiment, I am taking two controls i.e. only cells and cells + DHR and the Mean fluorescence intensity (MFI) are 200 and 4000 respectively. However,the MFI of the third tube containing Cells+DHR+PMA is 16000. I was wondering if it can be considered as positive for ROS measurement because the difference should be more between unstimulated and stimulated tube. So, can anyone help me with MFI values?
- Badges
- Science method