Introduction: Accurate measurement of multiple cytokines concurrently is essential for comprehensively understanding immune responses in various diseases. Goal: To develop a technological methodology for the simultaneous quantification of multiple cytokines in mouse serum using a flowcytometry Materials and Methods: Utilizing a C57BL/6 mouse model, we induced cytokine production by intraperitoneal injection of alpha-galactosylceramide (α-GalCer) and analyzed cytokine levels 24 hours later using a MACSQuant analyzer 10 equipped with LEGENDplex™ Mouse Th Panel (13-plex) assay. Means between groups were compared using the independent sample t test, and statistically significant differences were considered at p ≤ 0.05. Results: Our results demonstrate that the emitted intensity increased proportionally with standard concentration, with slightly elevated cytokine staining observed for MCP-1 and IL-10.
The concentration of each cytokine was determined using a regression equation based on the mean fluorescence intensity value of the cytokine in each standard. Importantly, we observed significantly highest levels of IFN-γ (p<0.05) in α-GalCer-injected mice, indicating successful induction of cytokine production, particularly from NKT cells. Notably, our investigation revealed that levels of IFN-b, IL-17A and IL-27 in α-GalCer-injected mice increased (p<0.05) those of other cytokines, compared to negative controls. The determination of multiple cytokines simultaneously via flowcytometry, facilitated by the LEGENDplex assay, holds significant importance in enabling comprehensive comparison of cytokine concentrations. Conclusion An experimental methodology for the simultaneous determination of multiple cytokines by flowcytometry was successfully developed.