Hi everyone, I differentiate THP-1 in macrophages with PMA 48h + 24h rest. After treatment with LPS I would expect an increase in ROS. But this increase compared to control I cannot get. I have tried different times 30', 4h, 12h, 24h. What do you think I'm doing wrong?
Hi Giulia!
ROS are very unstable. Maybe the times you have tried are too long (4h, 12h and 24h). I´d recommend you to try 30', 1h, 1,5h and 2h, not more than that. What is your positive control like? What do you use do stimulate ROS production in the positive control?
Hope my suggestion helps you!
Good luck!
Hi Angelica, thank you very much for your replying. I tried 30' but had no effect. I used LPS-treated macrophages as my positive control. Because I wanted to evaluate the immunomodulatory activity on macrophages. But in the past for other purposes I have also used TBHP 100 uM for 2h. Do you recommend anything else?
TBHP is fine. I have used it in my assays also, and it worked very well. I just treated my cells for 1h instead of 2h, but my cells were serum extracted leukocytes. When you used TBHP did your assay work?
If the TBHP assay worked, I´d recommend you to stop the differentiation assay for a while. Then, try the times I said before by using TBHP, so you can check if ROS are really being produced. By trying this you´ll check the ROS production, the detection method (which I think, you might be doing flow cytometry, is it right?) and if the kit is working. I think that at these times you might see the production increase little by little and even see if it reduces.
It´s a step behind of your assays, but then you will be able to carry on with no doubts.
Hope to have helped. Let me know if there is any other questions about and if it worked.
Good luck!
Thank you very much. You are really very kind. Yes I detect ROS with flow cytofluorimetry. On that subject, I also wanted to ask you another question. I would like to make sure that I perform the assay well. How do you perform it? I first pre-treat the cells with the antoxidant for 24h, then add TBHP for two hours. Finally I harvest the cells, do two washes and add the probe for 30 min at 37°C. I wash cells and acquire by FACs. Do you think I am doing something wrong in the procedure?
When I did this assay, I used to do all the procedures in the same day, as the kit recommended doing it in the same day. I'm going to attach the protocol I used, so it may help you. But what I used to do was: treat cells with antioxidant for 1h. Then, I added TBHP for another 1h and then added the probe for 1h. On the last 15 min of the probe incubation, I added the cell death marker for 15 mim. I didn´t washed cells, as the manufacturer recommended not doing it.
I don´t know what kit you are using or if you´re trying in-house method, but this protocol worked pretty well to me. If you have time, maybe you could try doing everything in one day and compare to your protocol. Or maybe, try not doing the washes: keep your original protocol, but just adding the reagents.
On the protocol I attached, I corrected the volumes for 200-300 uL, as you will see, the protocol final volume is 1mL.
Hope it works and you get your results!
