I'm doing a proliferation assay of human lymphocytes, but I would like to know if FBS may induce proliferation in controls. I´ll use RPMI-1640 medium to assay with FBS but, I think this serum can induce in vitro stimulus in human lymphocytes. I appreciate your contributions.
I have never done proliferation assays, but i have used stimulated lymphocytes to grow HIV virus in cell culture. We used FBS or FCS supplemented with PHA and IL-2 to activate lymphocytes and allow infection, serum alone would not allow infection of cells. Still, this is not a very sensitive method to detect activation and I guess there could still have been low-level stimulation unable to support efficient HIV infection.
If I were you I would set-up a series of controls with or without serum, and maybe experiment with some serum-free media.
I totally agree with Andrea. Controls are always sooo important. I think, that FBS won't activate lymphocytes, because it just mimiks the natural environment of your cells. Furthermore, I worked with gammadelta-T cells during my undergraduate studies and, as far as I can remember, we always used FBS in the medium. But it should be heat-inactivated, because proteins of the complement system can activate lymphocytes. There are several publications, dealing with this.
I´m planning to do a co-culture of this human lymphocytes with macrophages derived from THP-1 cells for immune assay. Do you think about if FBS can be used for this assay?
FBS or FCS can activate cells in proliferation assays. I don't know how you stimulate proliferation (mitogens, cytokines, allogeneic cells etc) anyway it is essential that you use controls (cells and medium +serum) and calculate the proliferation taking into account the background response due to the presence of serum proteins in the culture medium, Register the change of lots, the activation stimulus may change with different lots of serum.
It is true that FBS does cause non=specific stimulation, but can still be used as long as you can calculate this non-specific results and finalize your results. You neede to inactivate each time thelot of FBS is used.
Dr.H. N. Madhavan
You might try human AB serum if you are very concerned about non-specific stimulation with FBS.
Thanks Barbara, Do you think if it would be advisable to use autologous serum? Why human AB serum? Why i can´t use other type of human serum?
Hi Javier; Autologous serum (from normal individuals) would be good, but many times it may not be available, or because of disease state and/or treatment (from patients) it may not be a good experimental choice. Human AB helps to eliminate potential blood group reactions in the culture.
FBS does stimulate cell proliferation. If you intend to specifically understand the physiology of a specific stem cell, I advice you to use serum derived from the same person.
FBS does induce proliferation of cells. To understand or study the physiology of the stem cells yo are working, you have to use the serum derived from the same person from whom you derived the stem cells.
Hi Javier,
when I am giving lectures in cell and tissue culture, we do have a particular session about serum, FCS and others. In general, serum is a black box with so many ingredients, hormones, cytokines, growth factors but also antibodies (i.e. against growth hormone) which might and most probably will interfere with your cultures. So you could either try a serum substitute or even better, switch to defined serum-free media. We use them for neuronal and neural stem cell cultures, as well as for many other cell types. it its definitely a bit harder to work with, but worth the effort.
By the way, where is your university, in Merida? and does you name mean "jaguar" in the maya language?
Best
KH
Dear Javier,
you will even get background proliferation using autologous serum (see J.M. Moser et al. / Journal of Immunological Methods 353 (2010) 8–19). We are performing T cell proliferation assays in serum-free conditions using X-Vivo 15 (Lonza). But at the end you will do best by testing different conditions to establish your system and to see which condition gives you the best signal-to-noise ratio.
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