DCF Assay to detect oxidative stress - Fluorescence lower than expected - recommended improvements? (Mar/22/2010 )

Hi all,

I am currently trying to use the DCF assay in the flow cytometer. Although the results generally meet my expectations, the changes in fluorescence levels are considerably lower than I expected.

For those of you who are not familiar with the DCF assay, it's a method to measure the levels intracellular reactive oxigen species (ROS). It was first described for microplate reader in 1999 by Wang & Joseph, although the original method had already been discovered in the 60s. Basically, cells are incubated with the profluorescent, lipophilic H2-DCF-DA (dihydrodichlorofluorescein diacetate) which can diffuse through the cell membrane. Inside, the acetate groups are cleaved by cellular esterases so the resulting H2-DCF cannot leave the cells. Reaction with ROS, primarily hydrogen peroxide (H2O2), results in the fluorescent molecule DCF (max. emission ~ 530 nm), so that DCF fluorescence can be used as a measure for intacellular ROS levels.

I've use this method before on a microplate reader with some success, however I would like to try it on a flow cytometer now. I've read some papers on it, of course, so here is the preliminary protocol I've used. Recommendations for improvements are very welcome.

All media contained 10% FBS and 1% Pen/Strep, and all incubation steps were at 37°C and 5% CO2 if not stated otherwise.

- Grow 0.5 x 10^6 cells in 2 mL DMEM for 24 h (cell line Hct-116, adherent human colon carcinoma cells)
- Add testing substance (in this case 50 µM Fisetin) to medium, incubate for another 4 h
- Remove medium, rinse 1x with pre-warmed PBS, add 2 mL pre-warmed DMEM (without phenol red) containing 10 µM H2-DCF-DA; incubate for 30 min
- Remove medium, rinse 1x with pre-warmed PBS, add 2 mL pre-warmed DMEM (without phenol red) containing 250 µM H2O2; incubate for 60 min
- Remove medium, rinse 1x with pre-warmed PBS, harvest cells using 300 µL trypsin for 2-3 min at 37°C, then resuspend in 1 mL ice-cold PBS (Ca/Mg free) and keep on ice in the dark until measurement
- Measure fluorescence in the flow cytometer (Accuri C6, Accuri, Ann Arbor, MI, USA; Ex 488 nm; FL-1 530nm +/- 15; 10,000 events)

The substances I used were Fisetin, a flavonoid that is known to decrease intracellular ROS levels, H2O2 to induce oxidative stress, and the H2-DCF-DA probe. Now the mean DCF fluorescence as represented by the integrated pulse area for FL-1 (530 nm) for each of the six samples was as follows:

(- Fisetin, - H2-DCF-DA, - H2O2) 1838 (1.00) negative control
(+ Fisetin, - H2-DCF-DA, - H2O2) 1991 (1.08) Fisetin autofluorescence control
(- Fisetin, + H2-DCF-DA, - H2O2) 2151 (1.17) DCF / endogenous ROS control
(+ Fisetin, + H2-DCF-DA, - H2O2) 2297 (1.25) Fisetin ROS generation control
(- Fisetin, + H2-DCF-DA, + H2O2) 4674 (2.54) positive control
(+ Fisetin, + H2-DCF-DA, + H2O2) 3508 (1.91) actual sample

Although these results meet my expectations more or less, I would have expected a considerably higher increase in fluorescence levels in the positive control. In similar experiments in the microplate reader, the positive control yielded fluorescence levels up to 20 times those of the DCF control, in which no oxidative stress was induced so that the H2-DCF only reacted with the ROS naturally occuring in the cells. As you can see, here it was only about twice as high, and only about 2.5 times as high as in the negative control, which did not even contain the H2-DCF-DA probe.
Any ideas on what might be the reason for this?

Hi Aquifex! Sorry I don't have an answer to your question. I was just looking online for a protocol for DCF assay in a plate reader and your post came out as a result.
Will you be so kind to send me a detailed protocol for that, since you said it worked for you?
Thanks a lot, good luck with your flow cytometry

manchester on Mar 23 2010, 03:22 PM said:

Sure, although I'd recommend to check whether you can adapt it to the cells you use. The cell line I use is Hct-116, adherent human colon carcinoma cells. The protocol I used for the plate reader was very similar to the one described above and is actually pretty straightforward:

All media contained 10% FBS and 1% Pen/Strep, and all incubation steps were at 37°C and 5% CO2 if not stated otherwise.

1.) Grow 1 x 10^4 (10,000) cells in 100 µL DMEM per well in a 96 well plate for at least 24 h so they can attach properly
2.) Add in at to medium and incubate for . Make sure your substance and / or solvent is not toxic at the concentrations you use, or if it is, take this into account when evaluating the results
3.) Remove medium, carefully rinse 1x with pre-warmed PBS, add 100 µL pre-warmed DMEM (without phenol red) containing 10 µM H2-DCF-DA and incubate for 30 min in the dark
4.) Remove medium, carefully rinse 1x with pre-warmed PBS, add 100 µL pre-warmed DMEM (without phenol red) containing 250 µM H2O2 and IMMEDIATELY start measurement in the plate reader (this is important if you want to obtain a time curve for the increase in DCF fluorescence, as the H2-DCF starts reacting strongly with the H2O2 as soon as you add it. I recommend using a multi pipette or a multipette/stepper). I always measured for 1 h in 5 min intervals.

You should probably examine the cells under the light microscope after the measurement, but NOT before, as H2-DCF is supposed to be photosensitive. Also, as soon as you add the H2-DCF-DA, you should probably work in a dimmed room. Some people say this is crucial, other say H2-DCF-DA is not photosensitive at all - personally, I did not bother to find out who's right

The washing with PBS at step 3 may be left out if you have only few cells, but the washing at step 4 is essential. Otherwise there will be extracellular H2-DCF-DA left in the wells, which will react with the H2O2 and thus distort your results.

For the controls: I recommend using the same five controls I used above. The negative, positive and DCF control are obviously crucial. The control containing your test substance, but neither DCF nor H2O2 is only necessary if you suppose that your test substance might be autofluorescent. If your test substance is disolved in something else than water, such as DMSO for example, you should add a volume of solvent equal to the volume of your sample to each of your controls. If you suppose that your solvent might have an impact on ROS generation (I#ve heard that DMSO might, for example), it gets even more complicated, as you will need controls both with and without your solvent

My plates always contained every sample and every control at least in triplicate, i.e. 3 or more wells treated the same.

I hope you'll find this helpful. If you have any further questions, feel free to ask. Helps this topic to stay on top, too

-Aquifex-

Really helpful thanks. I'll give it a go next week and let you know. I am using HT-1080 cells, hopefully the conditions will be similar for my model... no one ever did this assay in my lab.
Cheers

Aquifex on Mar 23 2010, 02:42 PM said:

manchester on Mar 23 2010, 03:22 PM said:

Hi Aquifex! Sorry I don't have an answer to your question. I was just looking online for a protocol for DCF assay in a plate reader and your post came out as a result.
Will you be so kind to send me a detailed protocol for that, since you said it worked for you?
Thanks a lot, good luck with your flow cytometry

Sure, although I'd recommend to check whether you can adapt it to the cells you use. The cell line I use is Hct-116, adherent human colon carcinoma cells. The protocol I used for the plate reader was very similar to the one described above and is actually pretty straightforward:

All media contained 10% FBS and 1% Pen/Strep, and all incubation steps were at 37°C and 5% CO2 if not stated otherwise.

1.) Grow 1 x 10^4 (10,000) cells in 100 µL DMEM per well in a 96 well plate for at least 24 h so they can attach properly
2.) Add in at to medium and incubate for . Make sure your substance and / or solvent is not toxic at the concentrations you use, or if it is, take this into account when evaluating the results
3.) Remove medium, carefully rinse 1x with pre-warmed PBS, add 100 µL pre-warmed DMEM (without phenol red) containing 10 µM H2-DCF-DA and incubate for 30 min in the dark
4.) Remove medium, carefully rinse 1x with pre-warmed PBS, add 100 µL pre-warmed DMEM (without phenol red) containing 250 µM H2O2 and IMMEDIATELY start measurement in the plate reader (this is important if you want to obtain a time curve for the increase in DCF fluorescence, as the H2-DCF starts reacting strongly with the H2O2 as soon as you add it. I recommend using a multi pipette or a multipette/stepper). I always measured for 1 h in 5 min intervals.

You should probably examine the cells under the light microscope after the measurement, but NOT before, as H2-DCF is supposed to be photosensitive. Also, as soon as you add the H2-DCF-DA, you should probably work in a dimmed room. Some people say this is crucial, other say H2-DCF-DA is not photosensitive at all - personally, I did not bother to find out who's right

The washing with PBS at step 3 may be left out if you have only few cells, but the washing at step 4 is essential. Otherwise there will be extracellular H2-DCF-DA left in the wells, which will react with the H2O2 and thus distort your results.

For the controls: I recommend using the same five controls I used above. The negative, positive and DCF control are obviously crucial. The control containing your test substance, but neither DCF nor H2O2 is only necessary if you suppose that your test substance might be autofluorescent. If your test substance is disolved in something else than water, such as DMSO for example, you should add a volume of solvent equal to the volume of your sample to each of your controls. If you suppose that your solvent might have an impact on ROS generation (I#ve heard that DMSO might, for example), it gets even more complicated, as you will need controls both with and without your solvent

My plates always contained every sample and every control at least in triplicate, i.e. 3 or more wells treated the same.

I hope you'll find this helpful. If you have any further questions, feel free to ask. Helps this topic to stay on top, too

-manchester-

Another possible change from my protocol that just came to my mind: the amount of cells per well (in my case 10,000) depends on how fast your cells proliferate (Hct-116 divides roughly once every ~8 h). You should have ~ 70-90 % confluence at the time of measurement, so depending on your cell line, you might want to use more or less cells.

Some things I forgot: Excitation wavelength was 485 nm (blue), measured emission wavelength was 535 nm (green), and the light pulse duration was 0,1 second.

-Aquifex-

Thanks again.
I did it using 10K cells and it worked fine for me One little problem is that I use black 96-well plates, so I can't really tell how confluent the cells are after 24h... next time i am doing a clear plate as well as a control.
Your advices were really useful. I am doing it again next week trying different cells concentrations just to confirm the results.

Aquifex on Mar 24 2010, 11:22 AM said:

Another possible change from my protocol that just came to my mind: the amount of cells per well (in my case 10,000) depends on how fast your cells proliferate (Hct-116 divides roughly once every ~8 h). You should have ~ 70-90 % confluence at the time of measurement, so depending on your cell line, you might want to use more or less cells.

Some things I forgot: Excitation wavelength was 485 nm (blue), measured emission wavelength was 535 nm (green), and the light pulse duration was 0,1 second.

-manchester-

Hi, I just start to learn how to measure ROS levle by FACS, but my problem is my cell is GFP labbelled. Is there another alterative way to measue ROS by flow cytometry except FITC conjugated? DCF probe is green only?

-ttsweetie-

ttsweetie on Apr 10 2010, 06:56 PM said:

Hi, I just start to learn how to measure ROS levle by FACS, but my problem is my cell is GFP labbelled. Is there another alterative way to measue ROS by flow cytometry except FITC conjugated? DCF probe is green only?

DCF is a fluorescein derivate, which will always fluoresce green. Alternative ROS probes are Dihydrorhodamines (DHRs) and Dihydroethidium (DHE). However, these are mostly green as well. Additionally, DCF, DHR and DHE react primarily with different types of ROS. DCF is specially sensitive to H2O2, DHE to Superoxide (O2•-). I've read somewhere that Rhodamine B has an emission maximum at 580 nm when excited at 488, which might just be in the FL2 channel, so Dihydrorhodamine B might be what you are looking for. However, don't take my word for it, as I don't use Rhodamines and thus don't know too much about them.

-Aquifex-

Hi,
did you get the problem with DCFDA-FACS fixed? Couse I have a similar problem with this stuff right now. Normally I used to preload with DCFDA and radiate my cells prior to making a single cell suspension and FACS it. But for some closer looks I needed it loaded afterwards. So I radiated first and loaded with 12,5 µm DCFDA für 15 min at 37°C, rinsed it with PBS twice and made a FACSable single cell suspension. As postive control I used 0,1 mM H2O2 für 10 min, following two rinses and same loading protocoll. Even though I know that both are inducing a lot of ROS I detected lower amounts (up to a half) than in non treated controls - which could simply not be the case.
So right now I'm searching for the reason of this strange results. Maybe you can help me with this.
Greetings Katzastrophe

-Katzastrophe-