DCFDA ROS assay by Flow Cytometry (FACS) Protocol in detail

                

                      

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