Recently I have been working on ROS production studies using the dye CM-H2DCFDA. On live cells (astrocytes) the concentration of dye is 2.5uM and incubation for 30min. The problem is that the dye is not entered in all cells and its probably that the laser is generating a effect during the record, which is the best form to improve it? What is the most recommendable increase in dye concentration? The experiment is for each cell not in an 96 plate. Thanks
I detected ROS using dihydroethidium. It worked well. ROS level may be different in different cells and so they will stain differently with ROS detecting dye. And that is what we r expecting to detect in an experiment. Generally the dye should be washed away using a PBS solution before taking the reading. reading should be taken immediately after the desired incubation time.
Hi, Daniela.
1. Incubate the drug-treated cells with 0.5 mM of 2,7–dichlorofluorescin diacetate (DCF-DA, Sigma) in a serum-free medium for 15~20 min depending on the cell lines.
NB: This should be performed in CO2 incubator.
2. Wash the cells 2x with PBS briefly to remove excess amount of dye.
3. Add pre-incubated fresh growth medium.
4. Record images very quickly, then you switch over to bright field or block prolonged UV or laser exposures.
NB: This is one of many failures that the novice comes across).
You can do spectrofluorimetric measurement (you need specific black coated 96- well dish).
NB: For FACS analysis of the detached cells, stain the cells separately (one dish by one dish) because the dye and DMSO vehicle are toxic, causing cell death, leaking and re-staining of healthy cells. Lower concentration may help.
Ref: Azacytidine-induced chemosensitivity to doxorubicin in human breast cancer MCF7 cells (2017).
Hi
For measuring the intracellular ROS in individual cells.
seed the cells on cover-slip in 6-well plate and incubated overnight for attachment.
Next day cells were treated with fresh medium containing your drug on interest.
The cells were further incubated at 37°C for desired time. At the end of incubation, stain the cells with 40 μM 2',7'-dichlorofluorescein-diacetate (DCFHDA) dye in media for 30 min at 37c.
Excess dye remove by washing with 1X PBS. Cover-slip was mounted on glass slide and observed under a fluorescence microscope.
Hi Daniela,
I use to measure the amount of ROS production quantitatively in live cells. I use exactly the same reagent CM-H2DCFDA as you are using. For this I use HBSS as washing buffer and I am describing here the protocol for 96-well plate.
1. After complete drug treatment, wash cells with HBSS (room temperature) for 2 times.
2. Add 5μM CM-H2DCFDA (freshly prepared in HBSS) in each well and incubate for 30 min in dark CO2 incubator.
3.After 30 min, remove the reagent and add HBSS and measure the ROS production immediately by measuring the fluorescence.
During the process, plate should be protected from light by wrapping with aluminium foil. It is very simple and works well in RAW 264.7 macrophages, HepG2 cells and MCF-7 cells.
Good luck.
Nirmala Tilija Pun
Hi Nirmala. Could you please tell me the concentration of HBSS you used for the assay?
Thanks!!
Hello Swati Shikha ,
This is just a buffer and we can use it directly (no need to be worried about concentration). We usually use HBSS from Hyclone. You can go through the link below.
https://www.fishersci.com/shop/products/hyclone-hank-s-1x-balanced-salt-solutions-13/p-7058036.
Good luck.
Hi Nirmala,
Can you suggest me the concentration of DSMO you have used to prepare h2dcfda stock solution?
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