I'm currently treating nucleus pulposus (adherent cells in the intervertebral disc) with various concentrations of cytokines and analyze the ROS level induced. After harvesting cells with TrypLE from a 6 well plate, adding DCFH-DA at 4°C for 30 min and make single-cell suspension by vortexing, I loaded the samples on BD Acurri C6 plus and got these results:
Q: How should I avoid cells from dying and get enough live cells reasonably to represent the overall situation? Could it be anything wrong with harvesting adherent cells? BTW C6 has a bad reputation for doing cell analysis, should I use another flow cytometer?
Hi Dear, I think you should try single cell suspension stain at 37C rather than at 4C. Once staining is over, gently agitate the cells and don't wash the cells. Read directly on FACS system. Hope it may help you.
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