Principle
DCFDA or H2DCFDA (2, 7-dichlorofluorescin diacetate) dye diffuse into the cell and deacetylated by the intracellular esterases as non-fluorescent dye and its oxidized by ROS into green fluorescent compound. The less ROS less fluorescence and high ROS high fluorescence so it can be detected or pictured at excitation at 495nm, emission at 529nm.
Image Ref: https://www.cellbiolabs.com/reactive-oxygen-species-ros-assay
DCFDA Stock: 10mM in DMSO (Dye is light sensitive so maintain dark)
DCFDA working stock: 10uM of DCFDA in SERUM FREE culture media.
Note: Serum
free because, serum contains cellular esterases so that might break the dye
before it enter into the cells and affect the results bringing high background
Protocol:
Ø Grow your cells in 6cm or 10cm dish
or 6well plate, just grow enough cells for the FACS analysis and treat your
compounds and incubate according to your cells and compounds, for example in case of HT22
cells 5mM glutamate treatment generates ROS at 8-12hrs, mostly around 10hrs.
Ø After your compound incubation remove
the media and wash dishes with serum free media to remove the remaining
serum in the dish.
Ø Gently add 5mL (or enough to cover
cell surface) of DCFDA 10uM working stock solution into the dish and incubate in 37℃ for 30min in CO2 Incubator. Maintain
Dark condition from here onwards; try to reduce its direct exposure to the
light, minimum exposure is better.
Ø After 30 min, remove the dye
containing media and wash dishes with PBS and trypsinize your cells. (Trypsin
can also induce ROS, therefore, trypsinize for 2mins only). And neutralize trypsin
with FBS 10% media and harvest cells, centrifuge for 5min 1500rpm.
Note: Avoid harsh handling because
stress may generate ROS so....be gentle
Ø Wash the cell pellet with 2ml of
pre-warmed HBSS 1X buffer (with Ca and Mg) and pellet the cells. (PBS also should be fine
here)
Ø Again Mix with 1mL HBSS buffer and
keep the samples in ice and do FACS. using Annexin V FITC channel because
its green fluorescence.
My Experiment results ;
Media: DMEM high glucose, 10%FBS, 1% PS.
Day 0: HT 22 cells 500,000 cells per 10cm dish was inoculated
in 4 dish
Day1: After 20hrs growth the 2 dish were inoculated with
100uM CS11(my compound) and 10uM then incubated for 2hrs and then 5mM Glutamate
were added in two dish.
After 10hrs the samples were harvested and FACS was
performed following this protocol
Glutamate treated has more ROS than control so, you see a shift in the peak in FACS like in the pics
Troubleshooting: The things I tried before I got the above successful protocol,
I am writing this because, I do not want you to repeat the same mistakes as i did.....- Initially, after the compounds and glutamate treatment and I harvested the cells using trypsin and then incubated with 10uM DCFDA in PBS buffer, and did FACS but it didn't work out , the control and glutamate treatment both were similar, the harvesting the cells after PBS wash seems to quench the generated ROS in the cells and then I changed the buffer to HBSS instead of PBS, and repeated this again but didn't help.
- Then in my next try I tried (the above successful method,) washing the dish with Serum free media and added the DCFDA directly in the Cell DISH, in that way the generated ROS immediately react with DCFDA and makes fluorescent compound so the dye stays inside, so now all you got to do is harvest and measure the fluorescence to measure the ROS, even if any extra ROS generated during the trypsin harvest most of the dye would have been converted already and its not going to affect much. Hope you got it
If you have any questions regarding this protocol, let me know in the comments section.
You can download pdf file from the link below
https://drive.google.com/file/d/1bFsX4sfamhP6M_3WS5-6Prc7-Iury2M_/view?ths=true
