1 day, Cell seeding
2 day, Cell drug treatment
After 24 h, we measured ROS to this method.
1) PBS wash, twice
2) 1/2 Trypsin/EDTA treatment, 5 min
3) After inactivating 1/2TE with FBS10% media, 2500rpm/3 min centrifuge.
4) After media of supernatant, add DCFDA 5 uM in PBS in each sample.
* positive control tBHP 1 mM
5) After sealing the sample with foil, incubation 37ºC 20 min.
6) After 2500rpm/3min C.F, wash with PBS of 1ml.
7) After 2500rpm/3min C.F, add with PBS of 400 ul.
8) After Vortexing, measuring with flowcytometry.
I want to check my protocol of DCFDA assay.
Please, comment my question !
Thanks.
HI Daiha,
please compare your protocol with my FACS protocol.
-Cell seeding and drugs treatment
-Wash your plates with serum free media. (x1)
- Incubate at 37C incubator for 30min with 10uM DCFDA in serum free media. (DCFDA stock: 10mM). Note: Serum contains esterases which can give false results as DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’ –dichlorofluorescein (DCF).
- After 30 min, wash x1 with PBS and trypsinize your cells. (Trypsin can also induce ROS, therefore, trypsinize for 3-4 min).
- Neutralized the trypsin by adding 1X FBS (in PBS).
- Centrifuge for 5min 1500rpm.
- Wash x1 with PBS (while vortexing) and centrifuge for 5 min 1500rpm.
- Resuspend in 200-500ul PBS (while vortexing) and go to FACs (FL-1).
Careful, when you are using using Trypsin or FBS!
All the best!
HI Daiha,
please compare your protocol with my FACS protocol.
-Cell seeding and drugs treatment
-Wash your plates with serum free media. (x1)
- Incubate at 37C incubator for 30min with 10uM DCFDA in serum free media. (DCFDA stock: 10mM). Note: Serum contains esterases which can give false results as DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’ –dichlorofluorescein (DCF).
- After 30 min, wash x1 with PBS and trypsinize your cells. (Trypsin can also induce ROS, therefore, trypsinize for 3-4 min).
- Neutralized the trypsin by adding 1X FBS (in PBS).
- Centrifuge for 5min 1500rpm.
- Wash x1 with PBS (while vortexing) and centrifuge for 5 min 1500rpm.
- Resuspend in 200-500ul PBS (while vortexing) and go to FACs (FL-1).
Careful, when you are using using Trypsin or FBS!
All the best!
Hiiii Madhurendra Singh,
Which cell line did you use? Will 10uM concentration work for all the cell lines? or do we need to standardize it for different cell lines?
Swati sudhakar Sahoo
Definitely not! You should always test different DCFDA concentrations when you change cell line using untreated cells before using it for your experiment
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