Hello all,
I would like to ask whether you have experienced that IL-8 could induce ROS production from neutrophils without priming step?
I tested two times (treating neutrophils with IL-8 100 ng/ml) and got one positive and one negative result. I wonder if the variations were due to the donors from whom neutrophils were isolated.
Thank you.
IL-8 it can trigger definitely intracellular ROS. I am positive that the obtained differences were due to the differences in the mature or less mature circulating neutrophils from your donors. We had the same problem with chemotactic peptides. Gain more donors to see what will be the average of the response. Use donors with the same age group as you will have also differences between normal aged donors and young adults. Success in all your experiments!
Thank you so much for your helpful suggestion.
May I ask some more questions?
1. What is the difference in ROS production between young and aged donors that you observed?
2. What media you used for culturing neutrophils? RPMI + Fetal bovin serum or RPMI + Bovine serum albumin, and how was the concentrations of the serum?
Thank you very much!
Duy.
1.There is a difference - younger adults have higher ROS levels when triggered with chemotactic peptides in comparison to old healthy donors. You have to take into account that you it is a kind of physiological "exhaustion".
2.We are immediately using our neutrophils and keeping them for the experiment in HBSS without Ca2+/Mg2+. Ions stimulate neutrophils also. Do not use FBS or BSA because it will stimulate your cells as well. When we have tried cultivating them in RPMI 10% FCS the post-stimuli ROS response was faint due to high background. If you still need to keep them cultivated several hours use gelatin (0.5mg/mL).
You are very welcomed,
Monica
Thank you again Monica,
Your suggestions are so helpful.
All the best,
Duy