I'm trying to mesure total ROS production in bone marrow derived dendritic cells with H2DCF-DA (Invitrogen), but I having some problems.
After differenciated, I plate the cells to 1x10^6/ml and treat them for 18hs with LPS 100ng/ml or left untreated. Next, I stain the cells with the surface marker CD11c-APC and a Live/Dead dye, wash them with PBS and finally incubate with 10uM H2DCF-DA in PBS for 30 minutes at 37°C, protected from light. I wash the cells with cold PBS, resuspend them in PBS-FBS2% and aquire them inmediatelly in a BD FacsCantoII.
The problem is that my untreated cells show a much higher geometric mean that the LPS treated cells.
I've tried different protocols, including resuspending the cells in RPMI-FBS10% before adding the H2DCF-DA in PBS, but I didn't have any luck.
I would really appreciate any help or suggestions.
Hi Antonella, I had the same problem but with other compounds, later I found out that the ROS probe leaked out from the cells since the probe accumulates in the cytoplasm, and the treated cells show an increase in cell permeability reducing H2DCF-DA uptake, In my case, I solved the problem using propidium iodine which allowed me to separate the population with the cell membrane intact and I got better results
Hi Juan Fernado Cadavid Vargas , thank you so much for your answer!
I'm using the Live/Dead Near IR dye, that it's suposed to acumulate in cells with compromised membranes, but I'm going to try doing it with propidium iodine. Hope I'll get better results. Thank you again!
If the cells are healthy ( the cell membrane intact) PI won't get in, and the tagging mode doesn't depend on the accumulation within the cell, but the intercalation in the DNA strands. This is like double check on the cell viability.