Don't think the maximum number exists. Usually, it is recommended at least 5 times with MilliQ water. In my experience 5 is perfect, but you can extend the washing by few more times if you have a strong color on the walls. I don't think you will ever have transparent water in the differentiated samples (unless you wash away all AlizarinR completely :))

Alternatively, run control wells with the same wash numbers. Then use subtraction analysis, i.e., subtract the alizarin red reading of the controls from your experimentals to get a more accurate reading.

Thanks guys for your suggestions and input. After ~2 hours of continuous washing, the stain did not fully disappear. So, I decided to assay the plate anyway. I then normalised the fluorescent signal to that of the control and it was fine.

Best

While looking for best Alizarin Red Staining protocol for Osteogenic differentiation I found this interesting discussion. I also have Query in this line of process

How long we should expose Alizarin Red  to cell. What is the best Incubation time for Alizarin Red Staining?

If i am not wrong then we should use 2% solution (pH 4.2-4.5)

Thank you in advance

Dear Saurabh,

I follow the protocol from Bianco et al.(Postnatal skeletal stem cells. Methods Enzymol 2006;419:117-148).

I summary the protocol here:

The cell cultures are fixed with cold 70% ethanol for 1 h at RT, and incubated with Alizarin red S solution (2% in water, pH 4.1-4.3, adjusted with NaOH) for 30 min. Excess stain is removed by washing 4 times with water.

Sincerely