I just received blood from a patient. I plan on isolating PMN from them. I don't think I will be doing experiments using PMN that same day I isolate it. So I was wondering if PMN can be cultured with RPMI media? Also, what is your recommendation of storing these PMN if you don't use it?
You have to use them the day you isolate them, I think the longest we store them is about 4-5h. You can't culture PMN as they are terminally differentiated. While waiting to use them for experiments, we also keep them on ice; they are very sensitive to activation. The warmer they are and the longer they're stored the more likely they are to become stimulated and go through the respiratory burst.
I simply suggest you not to use the cells, especially if you plan to perform functional assays…
You have to use them the day you isolate them, I think the longest we store them is about 4-5h. You can't culture PMN as they are terminally differentiated. While waiting to use them for experiments, we also keep them on ice; they are very sensitive to activation. The warmer they are and the longer they're stored the more likely they are to become stimulated and go through the respiratory burst.
Peripheral blood PMNs have very short life span from 12-18hours [Ref:Hoffman, Hematology book]. So proceed with the experiment as soon as you isolate them.
In addition to all excellent comments mentioned above please be aware that different anti-coagulant(s) used in blood drawing may significantly influence the endpoint(s) of your experiments involving PMN. Also, in regard to PMN activation during isolation/storage (mentioned by Eric Patterson above)- to minimize this unwanted phenomenon you should always : 1) keep number of PMN centrifugation to a minimum, 2) use "non-sticky" plastic tubes, 3) re-suspend PMN pellet extremely gently, 4) keep PMN on ice, 5) avoid "warming/cooling" of your PMN stock. Under these conditions your PMN will be happy for 4-6 hrs even in simple buffers (e.g. PBS). Presence of Ca/Mg ions in the buffer will increase the risk of PMN aggregation over time. In any case, I would not recommend using PMN older that 6 hrs. Good luck!
In addition to the other suggestions, if a cold room (2-4°C) or chromatography fridge is available, you can put the blood sample onto a tilt-action shaker for overnight storage. Gentle tilting helps keep the PMN from aggregating.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology