Increased green autofluorescence is a marker for non-invasive prediction of H₂O₂-induced cell death and decreases in the intracellular ATP of HaCaT cells

Abstract

Since oxidative stress plays important pathological roles in numerous diseases, it is of both critical theoretical and clinical significance to search for the approaches for predicting oxidative damage. Cellular models have great value for studying oxidative damage, which would be significantly promoted if non-invasive approaches for predicting oxidative damage can be established without the need of exogenous probes. In our current study, we tested our hypothesis that changes of the autofluorescence (AF) of cells may be used for predicting oxidative cellular damage.Our study found that H 2 O 2 dose-dependently increased the green AF of HaCaT keratinocyte cell line at non-nuclear regions assessed at 1 hr or 3 hrs after the H 2 O 2 exposures, while H 2 O 2 did not affect the green AF of other cell types tested in our study, including PC 12 cells and BV2 microglia. We further found that the increases in the AF of HaCaT cells are highly correlated with the H 2 O 2 -induced increases in early-stage apoptosis, late-stage apoptosis and necrosis assessed at 18 hrs after the H 2 O 2 exposures, which are also negatively correlated with the intracellular ATP levels of the H 2 O 2 -treated cells assessed at 18 hrs after the H 2 O 2 exposures. Collectively, our study has suggested that increased AF may become the first endogenous marker for non-invasive prediction of oxidative damage selectively for such cell types as HaCaT cells. Compared with traditional approaches, our method may have significant value for studying oxidative damage of keratinocytes with significantly higher efficiency and lower cost.

Patient-derived cancer organoid tracking with wide-field one-photon redox imaging to assess treatment response

“…The lower ORR is likely due to increased flavin production (riboflavin, flavin mononucleotide, and FAD) during the cellular response to cytotoxic stress, suggesting the ORR [NAD(P)H/(FAD + NAD(P)H)] is sensitive to cell death. 73,74 Interestingly, we found that the mean ORR was independent of organoid area for the PCOs imaged in this study (area range: 0.02 to 0.9 mm 2 ). Larger PCOs exhibited higher mean FAD intensity values due to larger dead cores, but this was compensated by increased NAD(P)H levels at the periphery, which we hypothesize is due to the presence of more viable cell material.…”

“…S4 in the Supplemental Materials). 73,74 Conversely, the intensity of NAD(P)H and FAD increases and decreases, respectively, in the core compared to the rim of hollow organoids. This results in a high ORR in the core of hollow organoids, which do not appear to have an apoptotic core (Fig.…”

Patient-derived cancer organoid tracking with widefield one-photon redox imaging to assess treatment response

“…The copyright holder for this preprint this version posted December 21, 2020. ; https://doi.org/10.1101/2020.12.19.423617 doi: bioRxiv preprint mononucleotide, and FAD) during the cellular response to cytotoxic stress, suggesting the optical redox ratio [NAD(P)H/(FAD + NAD(P)H)] is sensitive to cell death. 58,59 Interestingly, we found that the mean optical redox ratio was independent of organoid area for the PCOs imaged in this study (area range: 0.02-0.9mm ). Larger PCOs exhibited higher mean FAD intensity values due to larger dead cores, but this was compensated by increased NAD(P)H levels at the periphery, Statistical techniques such as mixed-effect effect models can use these time-course data to account for organoid-level heterogeneity and provide improved sensitivity to drug response.…”