Optimization of cell density and LPS concentration for the evaluation of nitric oxide production on BV-2 in a Griess assay
- Author:
Nasim Karimi Hosseini
;
Shinsmon Jose
;
Sharmili Vidyadaran
;
Syafinaz Amin Nordin
- Publication Type:Journal Article
- Keywords:
Griess assay;
Nitric oxide;
BV-2 cells;
LPS concentration;
Cell density
- MeSH:
Nitric Oxide
- From:Malaysian Journal of Medicine and Health Sciences
2014;10(2):1-8
- CountryMalaysia
- Language:English
-
Abstract:
Introduction: Production of nitric oxide (NO) is one of the main responses elicited by a variety of
immune cells such as macrophages (e.g. microglia, resident macrophages of brain), during inflammation.
Evaluation of NO levels in the inflammatory milieu is considered important to the understanding of the
intensity of an immune response; and has been performed using different methods including the Griess
assay. To assay NO in culture, an appropriate number of cells are stimulated into an inflammatory phenotype.
Common stimuli include lipopolysaccharide (LPS), IFN-γ and TNF-α. However, overt stimulation could
cause cell cytotoxicity therefore an ideal concentration of LPS should be used. Objective: To set-up a
model of BV-2 cell activation that allows the assay of detectable levels of NO. Optimization of BV-2
microglia cell density and LPS concentrations after stimulation by bacterial lipopolysaccharide (LPS)
for the Griess assay is demonstrated in this study. Methods: BV-2 microglia were cultured at different
cell densities, and treated with LPS at three concentrations (1, 5, 10 µg/ml). NO production in culture
supernatants were then measured at 18, 24, 48 and 72 hours. Moreover, methyl tetrazolium assay (MTT)
was also performed to ensure that NO measurement is performed at no-cytotoxic concentrations of
LPS. Results and Conclusions: NO production follows a temporal pattern. The density of 25000 cells/
well was the ideal seeding density for NO evaluation in BV-2 cells. BV-2 stimulation by LPS is dose
dependent, and NO levels are increased proportional to the LPS concentration up to 1.0µg/ml, whereas
the higher LPS concentrations are associated with decreased cell viability may be caused by the high
toxic levels of LPS or NO. Although Griess assay has been commonly used by the scientists, however,
optimization of its parameters on BV-2 cells will be useful for the experiments which will be performed
on this particular cell line. The optimized pattern of Griess assay on BV-2 cells was achieved in this
study, hence easier and more practical for the future scientists to perform Griess assay on BV-2 cells.
- Full text:P020150604503454668332.pdf
