I want to quantify calcium with alizarin red and extract it with cetylpyridinium chloride (CPC). As I found different SOPs for that questions regarding the right buffer and wave length came up.
Sometimes CPC is dissolved in Na2PO4 (http://www.sciencedirect.com/science/article/pii/S0003269704001332) , sometimes in water (http://journal.chemistrycentral.com/content/6/1/18). What is your experience?
After extraction with CPC, some people do a centrifuge step, I couldnt find details about speed and/or time. What is it good for?
I also found people to measure at wave length between 490 to 595 nm.
If anyone has a SOP that works find I'd be glad if you could share it here as detailed as possible.
Thanks in advance!
Hello Dominik,
I am working on osteogenic differentiation and CPC extraction is a routine practice in our lab and in my settings it works very well. First of all there is no any requirement of centrifugation for alizarin extraction by CPC and we never do that. You should dissolve CPC in double distilled water because after that the required pH (7.4 or 7-8) is automatically set and you don't need to set pH of buffer but try to keep solution a little bit warm or at 37 because CPC precipitates if it gets cold. Try to prepare fresh every time and if u want to store than storage should be on room temp only.
Coming to the procedure. You can dissolve 350mg CPC in 10ml of water and put this volume according to your plate well's volume. Incubate t 37 for 2hrs if you want maximum extraction of alizarin from stained site. You can do quick extraction if you give a mild shaking to plate while incubating. At the end of incubation period, take the solution out in wells of 96 well plate and read at 405nm. I have standardised this optimum wavelength through absorbance scan method in Tecan 7500 pro so no need to worry about that.
Hope it helps dear..!
Thank you for your answear.
I dont think I can use the method you described since it requires to detach the cell layer for centrifugation steps. We work with 3D matrices where cells cant be detached from the surface.
Do you have experience with the CPC method?
Hello Dominik,
I am working on osteogenic differentiation and CPC extraction is a routine practice in our lab and in my settings it works very well. First of all there is no any requirement of centrifugation for alizarin extraction by CPC and we never do that. You should dissolve CPC in double distilled water because after that the required pH (7.4 or 7-8) is automatically set and you don't need to set pH of buffer but try to keep solution a little bit warm or at 37 because CPC precipitates if it gets cold. Try to prepare fresh every time and if u want to store than storage should be on room temp only.
Coming to the procedure. You can dissolve 350mg CPC in 10ml of water and put this volume according to your plate well's volume. Incubate t 37 for 2hrs if you want maximum extraction of alizarin from stained site. You can do quick extraction if you give a mild shaking to plate while incubating. At the end of incubation period, take the solution out in wells of 96 well plate and read at 405nm. I have standardised this optimum wavelength through absorbance scan method in Tecan 7500 pro so no need to worry about that.
Hope it helps dear..!
Hello Ajay, thanks a lot. I think this will help :)
Is there a reason why your CPC solution is not 10% ? Or is 350mg/10ml just the stock?
Dear Dominik,
350mg/10ml is final working concentration and you just need to put CPC in required volume of water and its ready to use on cells as such.
It was some 3 years back when I calculated this concentration. Vaguely, it was a recommended concentration either in some protocol or it was mentioned in product datasheet (Sigma). Exactly I have to go through my old copies..
For the time being, hope this information is sufficient to carry ut your experimental procedure smoothly..!
Cheers!
Hi, Dominik.
In our lab CPC is used at 10% in NaH2PO4 at a pH ca. 4 and it works just fine without undergoing centrifugation. As for the measurement, it is usually performed at 550 +/- 20 nm.
You need to be cautious if you are to perform the experiment in 3D matrices, specially if they are hydrogel-like, because of adsorption, ... issues. It is always better (if possible) degrading the sample to completion after incubation with Alizarin and previous to CPC-induced release and quantification to maximize Ca-AR complex recovery.
Hope it helps.
Dear Ajay kumar
Thank you very much. i was working on this issue . Can we fix the cells in 4%formaldehyde and store in the same solution for a week's time without staining using Alizarin red, as i am currently not having the CPS ready. Will that have any impact ? i will be getting CPC next week. Thanks in advance.
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