I want to measure neutrophils derived intracellular ROS using DCF-DA as fluorescent probe by mirco-plate reader. As neutrophils are suspension cells, they normally do not attach with culture dish, even in attached culture dish (used for HUVEC like cells). So, I need to collect cells in 2 ml tube to remove excess DCF-DA and after washing re-suspend to culture dish. The problem, is during the collection and washing process there might be chance of losing some cells which may cause result differences between control and treatment groups. Can anybody explain?
Hi. To measure DCF fluorescence cells, I prefer to do it in a Flow Cytometer or in a Fluorimeter, using a cuvette. You can see it in this paper:
Article Mangifera indica L. extract (Vimang) and its main polyphenol...
We used to get around this by doing the assay in round bottom 96 well plates. You plate the cells at the desired density and then incubate them with DCF containing medium. At the end of incubation, spin cells down (1500 RPM, 7 minutes), remove the DCF containing medium, add fresh medium/ PBS and spin again. Resuspend cells in PBS/phenol free medium and measure fluorescence. If you still have issues losing cells, switch to a V bottom 96 well plate.
Yes, I did. Sorry I assumed that you have a centrifuge that can spin both tubes and plates.If you don't, then there would be some loss for sure. Just be careful when transferring the cells to tubes, use tubes with round bottom (or V bottom, if they are available) and then spin long with high rpm so cells pellet nicely.