*3.1.1 Colorimetric quantitative ALP activity*


*Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term… DOI: http://dx.doi.org/10.5772/intechopen.100286*

	- 1.Aspirate or pipette out all culture medium from each well of the 6-well culture plate that contains induced or control cells to be tested.
	- 2.Rinse the cells twice with PBS. Add 200 μL of PBS to the side of each well, not to dislodge the cells. Aspirate off the PBS and re-rinse.
	- 3.Add 125 μL of 0.5 N HCl to each well.
	- 4.Scrape the cells off of the surface using a cell scraper and transfer the cells and HCl to a 1.5 mL polypropylene microcentrifuge tube with a tight fitting cap.
	- 5.Add an additional 125 μL of 0.5 N HCl to each cell to recover any cells remaining in the well, and transfer this to the appropriate tube.
	- 6.Samples may be capped tightly and stored at −20°C if they are not to be tested immediately.
	- 7.Extract the calcium from the cells by shaking the tubes on an orbital shaker for 4 hours at 4°C. If using frozen samples, allow extra time for samples to thaw.
	- 8.Centrifuge the sample tubes at 500 g for 2 minutes.
	- 9.Carefully collect the supernatant with extracted calcium, without disrupting the cell pellets, and transfer these to a new tube.
	- 10.Prepare a standard curve with the calcium standard and determine the amount of calcium in each control and osteogenesis-induced samples. Follow the instructions provided in the Stanbio Total Calcium LiquiColor® Procedure No. 0150 (Stanbio Laboratory).

PuraMatrix™ hydrogel (BD Biosciences) is a 16-amino acid synthetic peptide hydrogel composed of a repeating sequence of arginine, alanine, aspartate, and alanine (RADA16) [31], which is widely used for 3-D culture.


*Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term… DOI: http://dx.doi.org/10.5772/intechopen.100286*
