I am trying to test whether my stimuli could induce ROS production on leukocyte or not. I am using KG- cells.
1. Firstly, I coat the disk with BAM (Biocompatible anchor for membrane).
2. Then KG-1 cells are cultured on the BAM-coated disk. After 1.5 hour of incubation, cells are anchored on the surface of the disk.
3. The cells are pre-loaded with Amplite ROS Green in Assay Buffer according to the manufacture instruction. Incubation time is 1 hour.
4. The loaded cells are treated with stimuli. (I add the stimuli directly diluted in PBS 1X into each disk containing ROS Green in Assay Buffer, don't need to wash out before)
I am using H2O2 as a positive control. Negative control is untreated cells (a same amount of PBS 1X)
5. Finally, I use Fluorescence microscope to observe the result.
I haven seen the high intensity of fluorescence in all the samples including Negative control (untreated cells). I wonder that does anchoring on BAM initially induce ROS production?
Is there any problem with my protocol?
This is my first time to perform the experiment with ROS production on suspension cells. I am very grateful for all your comments.
Hey Lily,
So far your steps sound good to me. If your cells produce ROS, just because of the adherence to your BAM, it will be seen in your untreated control. So your "untreated cells" is only a negative control to your cells treated with stimuli. You also need cells, which are untreated, but were also not adhered to a BAM (for example just on the well bottom). These cells are usable to compare, if and how much ROS your cells will produce once mounted on the BAM. Moreover I would suggest PMA as a physiological positive control. It activates a lot in cells and also ROS production. H2O2 works only as a postive control for your probe, but gives you no information, if your cells are producing ROS by themselves.
Please feel free to ask anytime, if you have questions.
All the best and stay healthy,
Marc
Dear Marc,
Thank you so much for your advice!
I performed my experiment today one more time. My protocol has been changed a little bit.
I did not use BAM coated dish for this time.
I just seed the cells on the well bottom
Then the cells was incubated for 1 hour with ROS Green solution.
I used PMA as the positive control as you suggested (I used100nM for 15 minutes of incubation). And also use H2O2 (just to compare).
This time, I change to use micro-plate reader to observe the result.
The fluorescence intensity of untreated cells, my test compound-treated cells and even PMA-treated cells were almost the same. They are all high. I am really confusing why the fluorescence intensity was so high right after staining step. Do you think that it depends on cell type? I am using KG-1 cells (Human acute myeloid leukemia).
I agree with you that using H2O2 as the positive control does not make sense. I still want to compare. And in H2O2 treated cells, the intensity was 2 fold higher than the others.
I am so grateful for your advice! If you have any comment, please let me know.
The intensity of your fluoresence depends on the concentration of your dye solution, your number of cells seeded in the wells and the incubation time of the dye. But that is not really that dramatic. When the ROS levels in your cells did not differ, with or without treatment, than it could be that they take longer do produce ROS after your stimuli or PMA. In our hands immortalized cell culture lines did not produce any ROS beyond base line. We made bad experiences with any type of culture cell lines concerning ROS measurements. Ex vivo cells worked always fine.
Keep me up to the process and all the best,
Marc
In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".
In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).
If you like, please have a look at:
Functions of ROS in Macrophages and Antimicrobial Immunity
February 2021, Antioxidants 10(2):313
DOI: https://doi.org/10.3390/antiox10020313
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