Hi every one
I used alizarin red staining for determine the amount of vascular calcification (VC) in presence and absence of 4 extracts to detect their inhibitory effects on VC. The protocols that I use: first I washed the cells with PBS 4 times, after that fixed them using ethanol 70% and then washed them by ddH2O(3times). After that I added alizarin red S (40mM, pH4.2). After 5min I removed excess dye and washed cells 4 times with PBS. After washing cell using invert microscope, I added cetylpyridinium chloride 100mM (300µl) per each well and read the absorbance after 30min. "But unfortunately I couldn't found good results, because the absorbance of some wells in normal condition(not in BGP presence) have higher absorbance than Cells in calcification condition(BGP 10mM)!!!! And my positive control (Levamisole) has only 8% inhibition effect in 10µg/ml concentration.
Could you please tell me tips that correct my experiment?
Best regard
Hi
Did you paper filtered your alizarin red? Do you really need to add cetylpyridinium chloride 100mM (300µl)?
I did Alizarin Red staining on the human mesenchymal stem cells differentiated into osteogenic lineage and I never had any problem with it.
I usually fixed the cells with 4% Formalin or 95% Methanol for 15-20 mins. wash with PBS and stained with alizarin red for 5min at room temp. Remove stained and washed with distilled water. Make sure you filter the alizarin red staining before stained the cells. And make sure you get the right pH.
Hope it helps!
Cheers
Yanny
Dear Yanny Marliana,
thanks a lot for your response. I need to add cetylpyridinium chloride because i want to investigate inhibitory effects of my extracts quantitatively!!
when I saw the stained mono layer using invert microscope I found that my extracts have inhibitory effect, but i want to compare their effect with colorimetric assay. so i used cetylpyridinium chloride.
I didn't use shacking after each washing step, Can that be problematic?
and Is there any software to analyze pictures of inverted microscope for calcification?
thanks
Fatemeh
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