I am trying to find the ROS production in adherent cells after exposing them to stress causing agents, using flow cytometry. I am no sure how should I go about the sample preparation. Please help
Gentle trypsinization of cells should not affect your results (it is more damaging to membrane proteins than to anything else). Treat your adherent cells in 6-well plate with your stress-inducing agents or solvent control; wash cells with PBS; add full growth medium with ROS-detecting reagent (H2-DCFDA or DHE or hydro-Cy5); incubate in CO2 incubator for appropriate time (usually 20-30 min); wash cells with PBS; gently trypsinize (for minimum time), re-suspend cells in an appropriate buffer (e.g. autoMACS running buffer) and analyze by flow cytometry without delay. Use untreated cells not stained with the ROS dye to set PMT gain in flow cytometer. In this way you will trypsinize cells after their staining with ROS dye, which will decrease very remote chances that trypsinization of cells would affect detected ROS levels.
It is recommended to use Fluorimetric analysis for finding ROS in adherent cells/ cell lines. Treat cells with H2DCFDA for detecting nonspecific ROS.If you want to be more specific then treat your cells with 3'-(p-aminophenyl) fluorescein (APF)= to detect OH radical, ONOO-, -OCl anions. Dihydroethidium (DHE)- Used for superoxide detection.
Follow Molecular probe's protocol of H2DCFDA for cells staining for FACS. (Give mild trypsinization for dislodging your cells).
u can use 2,7-dichlorofluorescein diacetate for flow cytometry analysis of ROS, its usual and simple protocol. pls see the enclose
thank you.. So regarding the adherent cells, can I treat the cells with the DCFDA dye and then expose the cells to stress causing particles, then incubate for specific time and then can I just wash the cells in PBS and scrape them to form single cell solutions?
I think Scrapping the cells and over-trypsinization is itself a stress for a cells. This may give false positive result. Another option is plate these cells on coverslip, treat it with your treatment followed by live florescence microscopy.
Depending on your stress-treatment you could also start treating the cells and mainly 30min before your incubation time stops you add the DCFDA for staining. Afterwards you wash the cells twice, trypsinize them and are able to measure them.
In general I would recommend to use the Carboxy-H2DCFDA that occurs in cells.
Use CellROX (http://www.lifetechnologies.com/order/catalog/product/C10422) from LTI. Just add it 60 minutes prior to harvest, and harvest as usual. Be careful though, it is light sensitive...so try to do as much of it as you can in the dark.
Hi, we usually use Mitosox Red, DCFH or 1,2,3 Rhodamine. Just treat whit your stress agents the time that you need, and, more less 30 minutes with the fluoresence treatment. Then trypsinize the cells, (1x), but have controls without stress agents so you can subtract the fluorescence produced by the stress of trypsin, and controls without fluoresence agent so you can substract self fluorescence of the cells too.
You can detach cells with Accutase. It's gentler cell detachment method than Trypsin.
Hi,
if you don't want to stress cells by tripsinization or scrapping them from plate you can do this experiment on plates with optical bottom. After stress-treatment of cells, incubate them with DCFDA solution for 30 minutes, wash with PBS, then add PBS and read in plate reader (in 96-well plate). This method is fast and easy in comparison to flow cytometry and what is most imporatnt do not generate additional stress for cells. What is important you possibly will need higher DCFDA concentration than for flow cytometry.
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology