Dear Jyoti

Please clear first:could you obtain the calcification of osteoblasts normally ?

Thank you Paulo for your reply. Yes I agree this method is a relative quantification of mineralization but if you refer to paper entitled "An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction" by Gregory et. al. 2004, you would obtain standard curves for quantification by mineralization.

The first method is the CPC method described by you, but this method is not sensitive for concentrations below 0.25mM ARS, I have tried myself too.

The second method is the acetic acid method which according to the paper is sensitive for lower concentrations also, but I have repeatedly tried to optimize the protocol in my laboratory, but I am not able to get the colour change(to red) and hence cannot colorimetrically quantitate ARS in standard graph.

Dear Juliane, I have obtained a standard curve by the Cetylpyridinium chloride (CPC) method by using various concentrations of ARS stain, but only for concentrations higher than 0.25mM. I am too searching for a direct method of quantitation.

Ahad, I think calcification by osteoblasts is the same as mineralization, I hope I have answered your question correctly.

For Alizarin red you can dissolve 34.23 mg of alizarin red in distilled water. Each 0.1 ml contains 1 µmoles of alizarin red. based on this calculation dilute with distilled water for further dilution now you can get a standard curve

Thank u Ramajayam G for your reply, but how would you read the absorbance of concentrations as low as 0.05 M as they are not detected colorimetrically ?

Dear Jyoti, You can prepare a standard curve starting from a stock solution of 40 mM Alizarin Red. I was preparing a standard curve consisting of serial dilutions of Alizarin Red in CPC (1:1) with the following concentration: 0,625mM, 0,3185mM, 0,156mM, 0,078 mM, 0,039 mM, blank (10% CPC solution). I hope it has been helpful!!!!

Hi

I have used canine osteosarcoma cell lines kt-osa1 and kt-osa4 cell line. Intially I introduced them in osteogeneic diffrenetiation media and used alizarin red assay as you know that osteogeneic differentiation media leads them to have matrix proteins. Now it looks like that cells has taken alizarin red assay, but question is it cells or matrix have taken stain , Can any body tell me about that?

I prepared 2mM concentration of ARS in 10%CPC. Serial 2-fold dilutions of this in 10%CPC gave me 1, 0.5, 0.25, 0.125mM solutions. Then I measured the OD at 531nm (different than 405nm which is for just ARS in water) using a Wallac Multitask plate reader. I also found visual color difference in my solutions. Hope this helps.

As many scientist have already answered, I always used serial dilutions. Here are the concentrations I used:

Standard (Concentration of Alizarian Red S stain) Alizarian Red Stain Solubilization Solution

A 1 mg/ml 50 ul Stock Solution 950 ul

B 500 ug/ml 500 ul of A 500 ul

C 250 ug/ml 400 ul of B 500 ul

D 100 ug/ml 500 ul of C 600 ul

E 50 ug/ml 500 ul of D 500 ul

F 25 ug/ml 500 ul of E 500 ul

G 10 ug/ml 400 ul of F 600 ul

H 5 ug/ml 500 ul of G 500 ul

I 0 ug/ml 0 ul 500 ul