Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters.

Assessing the physiological role of H2O2 requires sensitive techniques to quantify H2O2 and antioxidants in live cells. Here, we present a protocol to assess the mitochondrial redox state and unconjugated bilirubin levels in intact live primary hepatocytes from obese mice. We described steps to quantify H2O2, GSSG/GSH, and bilirubin content in the mitochondrial matrix and the cytosol using the fluorescent reporters roGFP2-ORP1, GRX1-roGFP2, and UnaG, respectively. We detail hepatocyte isolation, plating, and transduction and live-cell imaging using a high-content imaging reader. For complete details on the use and execution of this protocol, please refer to Shum et al.1.

Mesenchymal stem/stromal cell quality control: validation of mixed lymphocyte reaction assay using flow cytometry according to ICH Q2(R1).

BACKGROUND: Mesenchymal stem/stromal cells (MSC) have immunomodulatory properties, studied in a wide range of diseases. Validated quality controls must confirm this activity in the context of clinical trials. This study presents a method's validation, assessing MSC's ability to inhibit lymphocyte proliferation, according to the ICH Q2 standard. METHODS: MSC were co-cultured with CellTrace™ Violet-labeled Peripheral blood mononuclear cells (PBMC) coming from a bank of ten donors, at seven different ratios for 7 days. Cell trace violet PBMC bank was validated in parallel. Flow cytometry analysis was used to obtain the division percentage of T cells. The percentage of inhibition of lymphocyte proliferation by MSC, for each ratio X, was calculated using the formula: Ratio × percentage of inhibition = (control percentage of division-ratio × percentage of division)/control percentage of division. The inhibition percentage of lymphocyte proliferation function of co-culture ratios was represented in a line graph. The corresponding area under the curve was calculated, representing MSC's ability to inhibit lymphocyte proliferation. RESULTS: Two cell trace violet PBMC banks were compared for bank validation. When compared using four different MSC samples coming each from a different donor, their area under the curve did not show any statistical differences and were correlated. Moreover, the stability of one cell trace violet PBMC bank was confirmed up to 509 days of storage. Analytical parameters were investigated for method validation. Analysis of repeatability and reproducibility respectively showed a standard deviation of 6.1% and 4.6%. The assay was robust regarding PBMC, as no statistical differences were found between inhibitory activities when testing three adjacent concentrations of PBMC. Still, attention is needed on MSC quantity as it can influence results. Linearity was evaluated: the percentage of inhibition of lymphocyte proliferation function of co-culture ratios was linear on the exploited range. Finally, the assay measurement range allowed to differentiate MSC presenting different inhibition activities. CONCLUSION: This quantification method displayed low analytical variability and no inter-bank variability of PBMC. However, MSC quantification should be checked before co-culture to reduce variability. Therefore, it could be used for the qualification of MSC batches' immunomodulatory activity.