Optimization of ROS Measurement in PANC-1 Cells
ABSTRACT
Objective:
Accurate measurement of reactive oxygen species (ROS) is essential for understanding oxidative stress-related cellular responses. Among the available detection methods, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) is widely used due to its sensitivity and ease of application. However, signal variability due to differences in incubation time can impact data reliability. This study aimed to optimize the incubation time of H₂DCFDA for accurate detection of intracellular ROS levels in PANC-1 cells using flow cytometry.
Materials and Methods:
PANC-1 cells were cultured under optimized conditions and incubated with 10 µM H₂DCFDA from 5 minutes to 90 minutes. Following incubation, cells were detached, washed, and analyzed using flow cytometry (FITC channel). Three independent biological replicates were performed for each time point.
Results:
A time-dependent increase in intracellular ROS fluorescence was observed. At 5 minutes, 19.79% of cells were ROS-positive, which increased from 30.65% to 65.70% until 25 minutes, respectively. After that point, ROS-signal was saturated and measured approximately 90-95%, like positive control. The strongest fluorescence signal, was detected at 30 minutes, indicating a peak in probe oxidation and ROS detection efficiency.
Conclusion:
This study demonstrates that H₂DCFDA provides reliable time-sensitive ROS detection in PANC-1 cells, with 30-minute incubation offering optimal signal intensity without additional chemical induction. However, variations in ROS dynamics among different cell types underscore the need for cell-specific optimization of assay conditions.
Keywords:
H2DCFDA,
ROS assay,
flow cytometry,
oxidative stress,
PANC-1.
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