Various Factors Affect Lipopolysaccharide Sensitization in Cell Cultures

Abstract

Commercially available lipopolysaccharide (LPS) is commonly used in research. Although protocols for its use are well established, we experienced a loss of LPS responsiveness in our cell cultures despite no obvious experimental changes. Our cell lines were stimulated with LPS and the media quantified for LPS responsiveness via an IL-8 ELISA. We discovered that the major cause of signal loss was differences in fetal bovine serum (FBS) formulation and concentration. One FBS formulation was notably better at eliciting an IL-8 signal than the second FBS, and 10% FBS in media was better at inducing LPS responsiveness than lower concentrations. We urge researchers to be aware of inherent variations in seemingly commonplace reagents as they may be unexpected sources of inconsistencies.

Transcriptional and Post-Translational Regulation of Junctional Adhesion Molecule-B (JAM-B) in Leukocytes under Inflammatory Stimuli

“…It is noteworthy that there is a mismatch between the data for the dose response and the time responses for both JAM-A ( Figure 3 A,D) and JAM-B ( Figure 3 B,E) despite the cells being treated at similar passage numbers. It is possible that media and FBS batch variations might have led to the dampening of the response, as observed in a previous study by Yang et al [ 26 ].…”

“…Blood samples were obtained from healthy volunteers (National Health Service Blood and Transplant; Addenbrooke’s Hospital, Cambridge University Hospital, Cambridge, UK) after ethical approval by the Faculty of Medicine and Health Sciences Research Ethics Committee, University of East Anglia, and the NHS Health Research Authority Ethics Committee. Peripheral blood mononuclear cells (PBMCs) were isolated according to the methodology described by Layhad et al (2018), where blood was layered over Histopaque-1077 (Sigma Aldrich, Gillingham, UK) and centrifuged at 1000× g for 25 min and the top buffy coat was collected [ 26 ]. PBMCs were further processed using anti-CD14+ magnetic beads (Miltenyi Biotech, Surrey, UK) to isolate CD14+ monocytes, as described by Day et al (2019) [ 27 ], or were transferred to T75 flasks to adhere for 2 h and cultured at 37 °C, 5% CO 2 in RPMI 1640 medium containing 2 mM L-glutamine and supplemented with 10% ( v / v ) FBS, 50 IU/mL penicillin and 50 µg/mL streptomycin.…”

“…PBMCs were further processed using anti-CD14+ magnetic beads (Miltenyi Biotech, Surrey, UK) to isolate CD14+ monocytes, as described by Day et al (2019) [ 27 ], or were transferred to T75 flasks to adhere for 2 h and cultured at 37 °C, 5% CO 2 in RPMI 1640 medium containing 2 mM L-glutamine and supplemented with 10% ( v / v ) FBS, 50 IU/mL penicillin and 50 µg/mL streptomycin. Granulocyte–macrophage colony-stimulating factor (rHuGM-CSF) (Peprotech, London, UK) was then added and the cells were incubated in the above conditions for 6 days, as described by Layhad et al (2018) [ 26 ].…”

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination

“…This suggests an interaction between the component(s) of the media and the LPS response in this particular assay. Indeed, fetal bovine serum (FBS), a common reagent for media supplementation, has been shown to alter LPS sensitivity in cell cultures [58] as well alter EV analyses [59]. Thus, we tested unconditioned media without FBS supplementation and found that nearly all treatments were comparable to the LPS control, thus implicating FBS as the component responsible for the cytokine suppression (Figure 5).…”

Cytokine Profile and Anti-Inflammatory Activity of a Standardized Conditioned Medium Obtained by Coculture of Monocytes and Mesenchymal Stromal Cells (PRS CK STORM)

“…Other products similar to this one successfully employs a like blending strategy to obtain a homogeneous product from very heterogeneous components [68]. Given the variability to LPS stimulation observed in the reactivity of PBMCs from each donor, using a standardized cell line, such as THP-1, would be of great advantage, allowing greater reproducibility [69,70].…”