Optimization of buffer solutions to analyze inflammatory cytokines in gingival crevicular fluid by multiplex flow cytometry

Abstract

Objective: the aim of this study was to test two buffer solutions in order to attain a reliable and reproducible analysis of inflammatory cytokines (IL-1β, IL-6, TNF-α, OPG, OPN and OC), in gingival crevicular fluid (GCF) by flow cytometry. Material and Methods: GCF samples from healthy volunteers were collected with perio-paper strips and diluted either in phosphate buffered saline (PBS) or Tris-HCl buffer, with and without protease inhibitors (PI). Cytokine immunoassays were carried out by flow cytometry (Luminex Xmap 200) generating standard curves. Results: standards curves generated with the use of phosphate-buffered saline (PBS) demonstrated best adjustment for cytokines IL-1ß, IL-6 and TNF- α levels, when using Tris-HCl (p<0.05). Conclusions: The use of PBS buffer with the addition of PI provided reliable measurements of inflammatory biomarkers in GCF samples of healthy volunteers. Key words:Curve fitting, flow cytometer, immunoassay buffer, crevicular fluid, cytokines.

The Modulatory Effect of Gender and Cytomegalovirus-Seropositivity on Circulating Inflammatory Factors and Cognitive Performance in Elderly Individuals

“…The most frequently applied quantification of cytokine levels using Enzyme-linked Immunosorbent Assay (ELISA) technique may sometimes not be sensitive enough, due to the “presence of naturally occurring biological inhibitors in circulation, which might interfere with the detection of the respective cytokine” [ 90 ]. Also, multiplex techniques and even the Cytometric Bead Array (CBA) Enhanced Sensitivity Flex Set used in our study “are primarily designed to accommodate the simultaneous measurement of several analytes, and therefore compromises are inevitably made for the individual analytes” [ 91 ]. Despite these limitations, results obtained in the present study for the most of the pro- and anti-inflammatory cytokines and other factors related to low-grade inflammation are rather consistent.…”

Evaluation of the reactive oxygen metabolite levels in plasma, gingival crevicular fluid, and saliva in generalized chronic periodontitis patients before and after nonsurgical periodontal therapy: A case–control and interventional study

“…PBS, being isotonic, increases the volume of GCF obtained without affecting the contents. [ 9 ] The samples were centrifuged to obtain the supernatant which was stored at -70°C until the assay. GCF collection using micropipettes has its own advantages and disadvantages.…”