Problem: Cells produce high amounts of IL6 followed by TNF and lower concentrations of IL10 within just hours from cell culture start point regardless of stimulation status (LPS vs. non-treated). We tried to deplete our medium to the point we used only fresh RPMI but it only lowered the level of IL10. Overnight resting of the cells followed by medium replacement didn’t solve the problem. Cytokine analysed: IL2, IL4, IL6, IL10, TNF, IFN-γ, IL17A.
PBMCs contamination with bacteria is unlikely as the cell culture was set every time from the beginning and workplace was decontaminated between every try. Antibiotic-antimycotic solution was used when full medium was used.
Methods: Human PBMCs were isolated and washed according to standard protocols from EDTA containing blood collection system using Histopaque1077. Cells are placed in 24-well plates in density of 5x10^5 cell per ml. Medium used – RPMI glutamax with standard supplementation (or without).
Hello Przemysław Wiktor Śliwka
High cytokine secretion of untreated PBMCs in vitro is possibly because of spontaneous in vitro release of cytokines due to prior in vivo stimulation. This is likely to happen in acutely ill and healthy persons living in an area where certain diseases are prevalent such as human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections.
Spontaneous cytokine production, albeit unusual in industrialized societies, can occur in some individuals and may not be that unusual in persons in developing countries. So, you need to obtain the history of the individual (ill or healthy persons) from whom you obtain these PBMCs to confirm the above finding.
You may want to refer to the articles attached below for more information on this subject. It will be helpful.
Article Spontaneous Cytokine Production and Its Effect on Induced Production
Article In vitro cytokine production by normal human peripheral bloo...
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