Thank you Mike. I had tried the Sigma kit but your sugestion to trypsinize
the cells first is shocking in its simplicity. Why didn't I think of it?
Thanks again, Donna

-----Original Message-----
From: Michael Archambault [mailto:marchambault@osiristx.com]
Sent: Monday, July 30, 2001 9:03 AM
To: 'Montague, Donna C'; 'Histonet'
Subject: RE: Molar ratio of Ca2+ for Alizarin stain


Hello Donna

Quantifying calcium is something we do on a routine basis from cultured
osteoblasts.  The easiest way we've found is to scrape up the cells in
dilute HCl (0.5N) and then quantify the calcium using a Sigma diagnostic kit
(sigma kit 587-A which you then read with a spectrophotometer), interpolate
from a calcium standard curve, and you can get a hard number.  We generally
work with 35mm culture dishes, which yield plenty of calcium to quantify.
The only caution- you will get all of the calcium in this manner-
osteoblasts like to deposit mineral onto the tissue culture surface, so
you'll be solubilizing and quantifying that as well.  To measure
intracellular calcium, we trypsinize the cells, and then solubilize them for
the assay.  This might be easier than trying to deal with aliziarin red in
solution.

-Mike

Michael Archambault, Research Scientist
Bone and Soft Tissue Program
Osiris Therapeutics Inc.
2001 Aliceanna St  Baltimore, MD 21231
410 522-5005 x 226
marchambault@osiristx.com


-----Original Message-----
From: Montague, Donna C [mailto:MontagueDonnaC@uams.edu]
Sent: Friday, July 27, 2001 2:31 PM
To: 'Histonet'
Subject: Molar ratio of Ca2+ for Alizarin stain


Dr. Kiernan or any interested other:

We are using Alizarin red stain as a colorimetric indicator of Ca++ in
cultured osteoblasts. I was thinking perhaps we could describe the extracted
and spectrophotometrically quantified alizarin (acidified and read at 450
nm) in terms of an equivalent molar concentration of calcium. Further, by
looking up the chemical structure in Conn's, I surmise the equivalence ratio
would be 1 mole Alizarin per 2 moles Calcium. Do you concur?

Donna Montague, M.S.
Research Associate
Physiology/Biophysics and Orthopae