[Histonet] Osteoclasts isolated from blood

From:"Hermina Borgerink"

Hello everyone,

I received the following e-mail request from a researcher in India.
However, not being familiar with the type of work she is doing, I
thought I would post her questions to the Histonet to see if anyone can
help her out.  

"I am working on expression of salmon calictonin in
E.coli and then assaying its action, for which i will
be isolating osteoclasts from pheripheral blood by
using Histopaque 1077, and M-Csf, etc.Then, i would
stain them with TRAP stain,as guided by you, for
osteoclasts isolating confirmation. Later, I would be
staining with touilidine blue, for analysing the
decrease in resorption pits with calcitonin
action(both number and radius). I need to enquire:
1.) whether i can do TRAP with osteoclasts isolated
from blood.
2.)Incubation period required for TRAP assay and
analysing resorption pits.
3.)Is it possible to keep osteoclasts in MEM with
Recombinant M_CSF, ODF, FBS,etc for 8-10 days ,during
the study, with out using any cell line."

Any input would be greatly appreciated.

Hermina M. Borgerink, BA, HTL(ASCP)QIHC
Wake Forest University Health Sciences
Department of Pathology
Medical Center Blvd.
Winston-Salem, NC 27157
Tel. (336) 716-1538
Fax (336) 716-1515
e-mail hborgeri@wfubmc.edu

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