title: Experimental and theoretical analysis of cytokine release for the study of radiation-induced bystander effect.
abstract: OBJECTIVE
  To clarify the experimental conditions that might influence the release of cytokines in the culture medium and give some basic input for building a model for cytokine (e.g., Interleukin-6, IL-6) regulation in the case of 'sham irradiation' and after ionising radiation exposure.
  
  
  METHODS
  The influence of cell type, cell density, medium volume, medium storage temperature and other methodological aspects on IL-6 and Interleukin-8 (IL-8) release were investigated. In addition, the effects over the time of different doses of gamma irradiation on the clonogenic survival of bystander cells and on the secretion of these cytokines were studied.
  
  
  RESULTS
  We observed significant decreases of clonogenic survival in AG01522 and T98G cells after the transfer of medium collected 5 and 20 h after low doses of gamma irradiation. Concerning the Interleukins' measurements, our experiments showed that the aggregate removal modalities tested, and up to 10 freeze-thaw cycles, do not have significant influence on the measurements of IL-6 concentration in the medium. We also observed that the IL-6 accumulated in the medium of human fibroblasts is not degraded when maintained at 37 degrees C. Sets of experiments demonstrated that cell density or medium volume do not influence the release of IL-6. On the contrary, our results showed that IL-8 released by glioblastoma cells strongly depends on the amount of medium. Finally, the exposure of fibroblasts to gamma irradiation has influence on the release kinetics of both IL-6 and IL-8 with peculiar features.
  
  
  CONCLUSIONS
  This study solved some of the methodological doubts concerning the study of bystander effects by means of the medium transfer technique; moreover it also highlighted some experimental aspects that need to be considered when approaching this sort of experiments.
authors.affiliation: Dipartimento di Fisica Nucleare e Teorica, Universita degli Studi di Pavia, Pavia, Italy. facoetti@unipv.it
authors.forename: Angelica
authors.initials: A
authors.lastname: Facoetti
authors.forename: Luca
authors.initials: L
authors.lastname: Mariotti
authors.forename: Francesca
authors.initials: F
authors.lastname: Ballarini
authors.forename: Alessia
authors.initials: A
authors.lastname: Bertolotti
authors.forename: Rosanna
authors.initials: R
authors.lastname: Nano
authors.forename: Francesca
authors.initials: F
authors.lastname: Pasi
authors.forename: Elena
authors.initials: E
authors.lastname: Ranza
authors.forename: Andrea
authors.initials: A
authors.lastname: Ottolenghi
mesh_terms: D024201:Bystander Effect / Q000528:radiation effects*; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis*; D016209:Interleukin-8 / Q000096:biosynthesis*
chemical_list: D015850:Interleukin-6; D016209:Interleukin-8