Hi, this is my first time trying this ROS experiment and I am not a biologist or familiar at all with cell stuff. So I would appreciate if anyone can break down this procedure step by step; that I was given to follow. And also clear up some of the jargons.
Protocol:
- Grow cells in 96 well culture plate , add the Nanoparticles with controls, Add the ROS detection reagent
(how many cells should I grow? 10,000? 5,000? - what should the control wells contain? PBS? - do I add ROS reagent right after adding Nanoparticles? or do I incubate for a period of time? & for how long?)
- Reconstitute the ROS indicator to make a concentrated stock solution. Keep tightly sealed until ready to use.
(what does it mean to "reconstitute" the ROS? - Do I use PBS to dissolve it? how much concentration?)
- Remove cells from growth media via pipetting.
(When do I remove cells? After adding Nanoparticles? After adding ROS? and after how long of incubation before I remove the cells?)
- Resuspend cells in pre-warmed buffer (PBS, HBSS, HEPES, or other simple physiological buffer) containing the probe to provide a final working concentration of ~1–10 µM dye.
(I plan on using PBS. Do I warm up the PBS? if so, for how long and what temperature? or does it mean to just use it at room temperature?. Also, does "containing the probe" mean contain the ROS? what "probe" are they talking about?)
- Incubate at the optimal temperature for the cells. Generally, a loading time of 5–60 minutes is sufficient.
(I have no idea what this means. Please explain. Does this mean I am incubating for 1 hour after adding the "PBS containing the probe"?)
- Remove the loading buffer; return the cells to prewarmed growth medium and incubate at the optimal temperature.
(Ok so I remove the "buffer containing the probe"?. But I don't understand what "return cells to prewarmed growth medium" means. and How do I know what the optimal temperature is?)
- Determine the baseline fluorescence intensity of a sample of the loaded cells prior to exposing the cells to Nanoparticles
(how do I do that?)
- Treat with Nanoparticles for 5 , 10 mins to 1 hr.
(Am I collecting samples? at different time points? - I don't understand this part either. Because I thought we removed the nanoparticles with the cells from growth media in the third bullet point)
- Extracellularly bound dye can be quenched using Trypan Blue (~0.0025%) in order to better distinguish the signal from the intracellular ROS response.
- Negative controls should be assessed as follows: Examine unstained cells for autofluorescence in the green emission range.
- Measure using excitation sources and filters appropriate for fluorescein Ex/Em: approx. 492 - 495/517–527nm
( I don't understand the last three bullet points either. I plan on using a 96 well plate because my microplate reader only works for 96 well plates)
Is there a way I can use a 6-well plate instead and use a fluorometer to obtain my data?
P.S I am using the U87MG cell line
Can anyone can please breakdown this protocol for me STEP by STEP?
Hello, I worked with U87MG in the past performing ROS assays. Please see the paper (DOI:10.2217/nnm-2017-0292). I'm gonna try to help as much as can.
First, seed 10,000 cells per well in medium overnight to allow cells to adhere to plate (mw96). Control wells should contain cells grown in medium. DCFDA probe can be incubated with cells before or after Np treatment. I usually add the probe after Np treatment. If the treatment with nanoparticles is for a short time (up to 2 - 4 hours), you can incubate with PBS. If the Np incubation time is longer (24 h) you need to use medium since PBS can be very stressful for your cells in long times.
DCFDA needs to be dissolved in organic solvents (DMSO, DMFA), please see data sheet for stock solution that you can aliquot and save at -20 for a few months. Afterwards, you need to bring to the final concentration with your cells in medium or PBS. The incubation with the probe (DCFDA) can be done for 30 min - 1 h.
You don´t need to remove your cells at least you performe flow cytometry. The medium with DCFDA needs to be removed and your attached cells washed with PBS. Put medium again to your cells in the wells and perform the lecture in a microplate reader Ex/Em: approx. 492 - 495/517–527nm. After the reading you could discount autofluorescence value from negative wells. Also, include a positive control (H2O2).
You can use a fluorometer but it is very laborious and time consuming. Try to find a fluorescence microplate reader or use flow cytometry.
Other works that you can read: DOI: 10.1016/s0891-5849(99)00107-0; 10.1007/978-1-60761-411-1_4
Hey Nina,
Luis already explained it very well. Only one suggestion. In the final medium (so when your cells go to the plate reader) you should not have any kind of serum or phenol red, because it highly influences and falsifies your fluorescence signal. You can use any kind of clear full medium. H2O2 as a postive control is good, but please do not (what I often read) add H2O2 to other wells, because you want to investigate, if your cells make ROS by themselves under the conditions you are testing.
For a detailed protocol for DCF staining and measurement in a plate reader, please also have a look at: DOI 10.1126/scisignal.aar5926
All the best and stay healthy,
Marc
Hey Nina,
The difficulty as always with ROS measurements is the questions: does your stimulus induce a quick or a slow ROS response or a ROS response at all? This means that we do not know, if and with what speed your cells might produce ROS after you add the stimulus (NPs). Moreover 10.000 cells might not be enough. I would also try 50.000 or 100.000. I send you a Plate exemaple for a 96er Well Plate how I would do it. Please use black non-clear bottom well plates, because in other plate types your fluorescence will be scattered 1. First of all you seed your cells in normal medium (which they like) with different cell numbers (marked as different grey shades in the plate scheme). In the rows A and H we normally do not seed cells, because the measurement fluctuations are very high. So we use row H for medium controls.
2. After seeding your cells, spin them down (450g, 5 min, 4°C)and let them adhere for 1h in your incubator.
3. After the hour remove the medium in all wells marked with "NP 24h" and "Only Medium with NP 24h" in the plate scheme.
4. Add new medium with Nanoparticels in the wells and leave the rest as it is. In these wells we will check, if your cells prodcue ROS after 24h incubation with NPs.
5. The next day, centrifuge your cells (450g for 5 mins 4°C), remove the medium from ALL wells.
6. Add 100 µl HBSS (PBS may be to harsh)to all wells to wash medium and NPs away .
7. Centrifuge again (450g, 5 min, 4°C)
8. During the centrifugation prepare the DCF staining solution. Add 20µl of DCF Stock solution into 9980 µl HBSS. Vortex and keep on ice.
9. Remove the HBSS.
10. Add 100 µl DCF staining solution to all wells (also in row H).
11. Incubate your cells for 30 min at 37°C (DCF gets in your cells, will be cleavead and can detect ROS).
12. After the incubation, centrifuge.
13. Remove the DCF solution from all wells
14. Add 100 µl HBSS to wash remaining DCF.
15. Centrifuge again.
16. Remove the HBSS.
17. Add 100 µl clear full medium (of your choice) in the wells labeld with "untreated cells, NP 24h, only Medium, only medium with NP 24h.
18. Add 100 µl clear full medium with H2O2 (I would try 50µM as final concentration) to wells labeled H2O2. This is the positive control for the DCF probe, but does not tell you if your cells are able to produce ROS on their own.
19. Add 100 µl clear full medium with NPs (at a concentration of your choice) to wells labeled with "NP freshly added". In these wells we can investiagate if your cells produce ROS right after they get stimulated with NPs.
20. Add 75 µl clear full medium to wells labeled with PMA. Add 25 µl PMA to all wells (final concentration 1ng/ml). This is our positive control for cellular ROS production. I never encoutered a cell, which does not prodcue ROS after PMA (at least after two hours of measurement). So if your cells do not produce ROS with PMA it is highly unlikely, that they will prodcue ROS with any stimulus.
21. Add 100 µl clear full medium to all wells to fill them up to a final volume of 200 µl per well.
22. Place the plate in a pre-heated (37°C) plate reader and measure every minute for two hours.
I attached the plate scheme and our manuscript.
A word of warning. First of all DCFDA is termed as intracellular ROS probe, but after cleavage in the cytosol it is highly diffusable. So you measure "Overall ROS" in the cell, not only intracellular. Second, we tried to measure ROS in different non-immune cells and it was quite hard, because these cells to not produce big amounts of ROS. Even worse are immortalized cell culture lines of any kind we tested. We tried RAW (macrophage), BV-2 (microglia) and HeLa. And non of them produced any kind of ROS, while their ex vivo isolated counterparts made a lot of ROS with the same stimulus. So you might get disappointed there.
I hope that helps you. Please ask any further question.
All the best, Marc
- Badges
- Science topic
- Similar topics
- Chemistry
- Nanotechnology
- Nanostructured Materials
- Nanoparticles