Hi all,
I'm currently incubating neutrophils for 6hrs, unstimulated and with IFN alpha, beta, gamma and gmcsf. I'm getting a lot of apoptosis (though less in my conditions) but this has been improved with 10% FCS. however its still ~47% apoptotic in my unstimulated. I had a couple of other issues with the incubation with more FCS, but I was wondering if I added 15-20% would it make a difference/improve?
Sophie
Dear Sophie, 1) obviously, neutrophils are the cells with very short lifespan (many of them less than 2 h). So, high rates of neutrophil death in several hours after isolation is their normal feature. Their survival can be extended by several stimuli, such as cytokines (IFNgamma) and agonists of TLR2 and TLR4. 2) Low concentration of FCS (less than 10%) is not optimal for different types of mammals' cells cultured in vitro, because insufficient concentration of protein in the medium results in cell swelling and can induce cell death. As a rule, rang of FCS concentrations between 10 and 20% is sufficient for successful establishing of primary cell cultures.
Dear both,
thank you for the advice. I was aiming for 15%. The experiment I am conducting is to look at GMCSF, and IFNs alpha, beta and gamma, and all are saving. Obviously I just don't want all my neutrophils to die before I can measure anything, and 4hrs I've found to be quite inconsistent
Sophie
Sophie, FCS contains so many factors that could activate PMN and then trigger cell death, I would probably avoid it for your study. You would probably be much better off with lipid free, endotoxin negative BSA instead. I suggest using BSA in the range of 0.1 to 1%. Note that adding BSA will acidify your buffer so you should adjust the pH back to the neutral range before adding cells.
Hi Robert,
Yes that is a factor that I have considered. However, I have been informed that the FCS is heat activated thus for my experiment, it shouldn't effect the cells too much. However, Ideas like this is really useful for future experiments
Yes, heat inactivation of FCS is essential in most experiments with live cultured cells. However heat inactivation mainly destroys C5a and a few other components of the complement cascade leaving the remainder of the cascade intact. Also, heat inactivation will not remove any growth factors lIke EGF, PDGF, transferrin, etc., nor will it remove IgG, IgM, hemoglobin, numerous cell attachment factors or truly huge amounts of fibronectin. You may want to do some reading about the composition of heat inactivated FCS and consider whether it might be a problem in your system. Unfortunately, in the presence of all these stimulants for neutrophil activation and apoptosis, the effects of your agonists of interest will very likely be completely masked by factors present in FCS.
Thanks for the advice Robert, that's quite interesting. I have previously used AB serum in the past, but I have been reluctant to use it at the moment due to the high numbers of neutrophils and conditions I need (we don't typically stock it as I'm the first person to be doing my experiments). However, in your experience would you consider this better or worse than FCS? (I have found it to be quite good but considering I'm using cytokines, I haven't used it currently for the same reasons you addressed before)
Sophie, AB serum may present the same problems as FCS. Your best bet might be tissue culture medium like DMEM or F12 without serum but with 0.1-1% BSA instead. These media usually contain bicarbonate which the PMN will like, but bicarbonate means that you will need to do your incubation in a CO2 incubator to keep the pH balanced. If you don't want to use an incubator then use a tissue culture medium w HEPES or another buffering system that will work properly in room air, but I would steer clear of any kind of serum or plasma for the type of expts you want to do. Serum or plasma will create such a complex system that your data may end up being uninterpretible.
I agree with Robert's comments.
Serum will activate neutrophils with subtle events such as calcium fluxes followed by F-actin polymerisation occurring within minutes of exposure. A commercial medium supplemented with low concentration of BSA sounds like a sensible option. I have previously maintained neutrophils in suspension for up to an hour using just Hepes buffered HBSS (as a negative control) with good results. Biol Cell. 1993;79(3):251-7.
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