Guava incyte version 3

Cells seeded at a density of 1 × 10⁵ cells/well in six-well plates were incubated with 10 μM DCFH₂-DA for 30 min at 4 °C while kept from light. Then, the cells were washed with PBS and pre-treated either with 5 mM NAC, 20 mg/mL sericin hydrolysates or 20 mg/mL unhydrolysed sericin for 60 min prior to exposure to 1 mM H₂O₂. After 30 min of treatment with H₂O₂, the cells were resuspended in PBS and immediately subjected to flow cytometry using Guava easyCyte benchtop flow cytometers (EMD Millipore, Darmstadt, Germany) for measurement of cellular fluorescence intensity of DCF at excitation/emission wavelengths of 488/538 nm. Cellular ROS level was a relative value of mean fluorescence intensity quantified by Guava InCyte version 3.1 software (EMD Millipore) between specific treatment and untreated control cells.

The level of intracellular ROS, O₂^·−, and OH^· were determined via flow cytometry using the fluorescence probe DCFH₂-DA, DHE, and HPF, respectively. For this, H460 cells at density of 3 × 10⁵ cells/well were cultured overnight in a 6-well plate, then pre-incubated with 10 µM of either DCFH₂-DA, DHE, or HPF for 30 min at 4 °C in the dark before further culturing with 50 µM cisplatin for 1–6 h. The cells were then washed and resuspended in PBS prior to immediately analyzing for fluorescence intensity using a Guava easyCyte Benchtop Flow Cytometer (EMD Millipore, Darmstadt, Germany) at an excitation/emission wavelength of 488/538, 488/610, and 490/515 nm for the detection of DCF, DHE, and HPF fluorescence intensity, respectively. The mean fluorescence intensity was quantified using the Guava InCyte version 3.1 software (EMD Millipore). The relative ROS level was derived from the fluorescence intensity ratio at the specific time point to that at 0 h.