ROS assay - (Aug/07/2011 )
hi all
iam do ROS assay in one of in vitro study but our absorbace data that read with plate reader is not significant. in other word in our data shown Fluorescence absorbance and according to our study our data shoud be additive but in our measurement data are erratic.
as regards i am use than DMEM containing phenol red but other cases are according to (PC12 cells were seeded in 96-well plates were incubated with increasing
concentrations of 6-OHDA and for 24 h. Cells were incubated with 5 mM DCFH-DA at 37 8C for 30 min, then washed twice with PBS.Finally the fluorescence
intensity of DCF was measured in a microplate-reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm),do you think our problem is due to use than this DMEM or due to other cases do you not help me?
sincerely
hamze on Sun Aug 7 20:05:23 2011 said:
hi all
iam do ROS assay in one of in vitro study but our absorbace data that read with plate reader is not significant. in other word in our data shown Fluorescence absorbance and according to our study our data shoud be additive but in our measurement data are erratic.
as regards i am use than DMEM containing phenol red but other cases are according to (PC12 cells were seeded in 96-well plates were incubated with increasing
concentrations of 6-OHDA and for 24 h. Cells were incubated with 5 mM DCFH-DA at 37 8C for 30 min, then washed twice with PBS.Finally the fluorescence
intensity of DCF was measured in a microplate-reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm),do you think our problem is due to use than this DMEM or due to other cases do you not help me?
sincerely
Hi Hamze,
You strictly have to perform your assay in a medium free of primary or secondary amine to avoid extracellular hydrolysis of your probe. Use PBS or HBSS. It's also better to make a first loading step where you incubate your cells with the probe, then wash and finally make your stimulation step before reading the fluorescence. In my hand, i found better sensitivity with a flow cytometer than a plate microreader for this assay.
Hi dear
thanks about your reply
about use than pbs i shoud say iam culture our cells in DMEM high glucose containing horse serum and FBS after 24 hours than treatment iam addithin DCF-DA in same medium and after 30 min washing whit PBS
about use than flow cytometer itis not avilable
sincerely