ROS assay using CM-H2DCFDA - (Dec/23/2009 )
I'm just wondering if there is anyone with experience using CM-H2DCFDA for the detection of reactive oxygen species that could answer some questions? My work is in the field of diabetes and I need to measure endogenous ROS islets (a cluster of cells) as well as after certain treatments (e.g. H2O2). I'll be measuring the DCF fluorescence using a fluorescence plate reader (FLUOstar Optima) rather than microscopy or flow cytometry. The problem I face is that islets are in suspension in the media, hence I will need to adapt the ROS assay protocols made for cell monolayers. Here are my questions:
1/ Do you add the probe to the cells before or after the treatment regime? I have been told by an expert in ROS assays with CM-H2DCFDA that you must always add the probe after the treatment as "dead cells don't produce ROS" and hence if the probe is already present inside the cells then the ROS accumulation in the dead cells will be captured. On the other hand, I have read on this forum that because cells don't "like" the probe they will spit it out and so it is better to add the probe just prior to measuring fluorescence in the plate reader.
2/Will sonication of cells after probe addition dramatically increase your signal? I ask this because I'm unsure whether you can get an accurate readout from an intact cluster of cells like the islet.
3/ Does anyone have an understanding of gain adjustment with regards to fluorescence readings?
Hopefully there will be some good advice coming my way.
I´m working with the probe to mesure ROS in human fibroblast cells (so adherent). I add the probe before the treatment. First I add the probe (diluited in PBS) prapared the same day. After the incubation, I add the treatment and mesure with a fluorescence plate reader. I´ve never worked with cells in suspensions. If you want to study the formation of ROS along the time with or without a treatment i´d add the treatment after the probe and doing a kinetic curve (for example one measure every five minutes). For example, after the probe, you can oxidize the cells with hydrogen peroxide and with hydrogen peroxide + treatment (or with the cells pretreated) and mesure the production or ROS. If your treatment works you can see minor production of ROS.
With cells in suspensions, the fluorescence obtained in a plate reader is good. But with cells in suspension I don´t know. If the number of cells is great i think you´ll obtein sufficient fluorescence to use a microplate reader.
I use the probe of invitrogen. If you need the data sheet please tell me.
Unfortunately the questions you ask can really only be answered by performing the experiments. I would also recommend contacting Invitrogen or Molecular probes, as they may have some suggestiongs for suspended cells...here is also the manual if you did not have it http://probes.invitrogen.com/media/pis/mp36103.pdf.
From my experience upon loading dyes into new cell types, you really just need to test it doing it both ways, that is before and after treatment, as well as using time courses - i.e. pretreat from 5, 15, 30, and 60 minutes. You can just use H2O2 or phorbal esters to produce ROS as a positive control. Some cells take up dye readily and retain it, while others have an active transport system that removes the dye quickly. If there is no published literature on islets, then you will need to determine it yourself. Also, like IKER said, set up a kinetic time course that takes readings every 10-20 seconds for 30-60 minutes for the fist couple of times, so that you know when you can stop reading. But again, IVGN may be able to help with suggestions.
Also, here is an application note from BMG LABTECH on the FLUOSTAR OPTIMA that talks about this same application in fungi, which I believe are non-adherent cells: http://www.bmglabtech.com/db_assets/applic...ce-FLUOstar.pdf.
As far as sonication, I would NOT recommend this because it can disrupt the cell membrane and it produces heat that can kill the cells, so you have to put the cells on ice or in a water bath. I would recommend a shaking protocol on the instrument for 8-20 seconds before you read the plate or before you activate them. This will help disperse clumps of cells.
As for gain adjustment, you can do it two ways: 1) if you have a positive control, set the gain to 90% of the positive control. By doing this, you are telling the instrument to set that well to 90% of the total RFUs of the instrument. The problem with this is that one of your samples may be higher than your positive control and may get maxed out. You can either than lower it to 80% of positive control, or you can do the second way. 2) you can set the gain to 10% of your blank, that is cells with no dye. Here you are doing the opposite, you are setting your blank to 10% of the total RFUs and theoretically eveything else should be higher. The problem with this is that your highest signal may get maxed out, but more likely your positive signal will only be a fraction of the total dynamic range of the instrument, therefore, your assay window will be smaller than if you set the gain on your postive controls. Try both to see which gives you the best S:B ratio.
Gain requirement! Once, though, you find a gain that works, you will want to use that same exact gain each time so that you can compare your results from plate to plate. If you have a different gain on each plate, you can NOT compare plates.
Hope this helps! Have fun!
Sorry for the delayed response. I want to thank you both for your detailed responses, as I am very glad for any help and advice that comes my way. I'm in the process of performing trial ROS assays to optimise/fine tune key steps. I will let you guys know how my experiments go!