How to release Ca2+ from calcified tissue? - (Dec/07/2008 )

Hi,

I have differentiated osteoblasts and would like to quantify the amount of Ca2+ deposited in the mineralized matrix. How should I release the Ca2+ from the tissue if I'm planning to measure the solubilized Ca2+ spectrophotometrically using o-cresolphthalein reaction?

Thanks.

Hi there,

we quantify Ca2+ with the Calcium CPC liquicolor kit from Stanbio Laboratories. Kit

We wash the cells, incubate them 1 min in 0,5 ml 0,5 M HCl, scrape cells in the HC, transfer them to a tube, add 0,5 ml Hcl to wash plate, then solubilize over night on a shaker and then use the kit with the included calcium standard to determine amount of Ca2+.

Stardust

Hi Stardust,

Thank you for your reply. I'm actually trying out the same kit from Stanbio, but am getting relatively low concentrations (~5mg/dL; I simply add 1 ml 0.6M HCl/well in a 6-well plate and incubate overnight at 4oC). May I ask a few more questions?

1. What cultures are you working with and what concentrations do you get?
2. Do you incubate overnight at 4oC?

Thanks!

Hi there,

I'm getting concentrations between 4 and 12 mg/dL, depending on the experiment.

I scrape the cells from the plate into HCl, then incubate the tubes with the detached cells at4 °C over night on a shaker

We are working with stem cells after osteogenic differentiation.

As I said, I incubate scraped of cells ON at 4°C

Stardust