Muse benchtop flow cytometer

Cell cycle assays were performed to examine the percentage of haploid cells in differentiating spermatogenic cultures. Cell cycle analyses were conducted using the Muse® Cell Cycle Assay Kit (EMD Millipore, Billerica, MA) as per manufacturer’s instructions. 10,000 events were analyzed for each treatment and the percentage of haploid cells calculated. Samples were run on the Muse® benchtop flow cytometer (EMD Millipore, Billerica, MA). Measurements of ROS were also conducted using a flow cytometry based approach. ROS generation was assessed using the Muse® Oxidative Stress Kit (EMD Millipore, Billerica, MA) as per manufacturer’s instructions. This kit determines the percentage of cells that are negative for ROS (healthy cells) and positive for ROS (cells exhibiting ROS). 10,000 events were analyzed for each treatment, and samples were run on the Muse® benchtop flow cytometer (EMD Millipore, Billerica, MA). % ROS+ was averaged from three separate experiments. Statistical significance was evaluated using paired student’s t test, p < 0.01.

ROS generation was assessed by the Muse® Oxidative Stress Kit (MilliporeSigma, Billerica, MA) by staining unfixed cells with dihydroethidium as per manufacturer’s instructions to prepare samples for flow cytometry. Samples were run on the Muse® benchtop flow cytometer (MilliporeSigma, Billerica, MA). For each flow cytometry-based experiment, 5,000 events were analyzed for five replications (n = 5) per chemical concentration and DMSO-only control.

Mitochondrial membrane potential was assessed using the Muse® MitoPotential Kit (MilliporeSigma, Billerica, MA) by staining unfixed cells with a supplied cationic, lipophilic dye and 7-AAD as per manufacturer’s instructions to prepare samples for flow cytometry. Samples were run on the Muse® benchtop flow cytometer (MilliporeSigma, Billerica, MA). For each flow cytometry-based experiment, 5,000 events were analyzed for four replications (n = 4) per chemical concentration and DMSO-only control.