Human smooth muscle cells characterization

Joanne Lannigan joannelannigan at virginia.edu
Wed Nov 2 12:14:04 EST 2005


Hi Yael:
I forwarded your question to one of our investigators who routinely stains
for alpha-SMA and here is his response:

"This antibody is a very good one.  We get a high level of specificity from 
the 1A4 clone, although we use the sigma-fitc conjugated one - 
Sigma#F3777.  This antibody is widely used by pathologists for assessing 
leimyosarcoma and SMC tumors.  However, at very high concentrations we've 
seen the antibody start to stain non-muscle actins.  I would suggest he do a
titration, we use 
a concentration of 1:1000.

If you notice in my cells, the actin only co-stains with the SMMHC, which is
a good internal control, and with our immunofluorescence of tissue sections
we see a high degree of specificity with the antibody.

If he is using a secondary antibody he may be experiencing nonspecificity
from the secondary.  If not he should try a lower concentration and 1-2 10
minute washes after primary fixation."

Hope this is helpful.

Joanne Lannigan, MS
Director, Flow Cytometry Core Facility
University of Virginia
Jordan Hall, Room 7067
P.O. Box 800734
Charlottesville, VA 22908-0734
Office: 434-924-0274
Lab: 434-243-2695
Fax: 434-982-1071
email: joannelannigan at virginia.edu
-----Original Message-----
From: Yael Sarnatzki [mailto:y.sarnatzki at mgvs.co.il] 
Sent: Monday, October 31, 2005 3:37 AM
To: cyto-inbox
Subject: Human smooth muscle cells characterization



Dear flowers,

My name is Yael and I work in an Israeli company called MGVS, a
cardiovascular cell and gene therapy company.
As part of our process, we isolate human saphenous vein smooth muscle cells
(hSVSMC).
These days we are trying to calibrate a method for identification of human
smooth muscle cells by alpha-SMA. Has anyone tried to characterize human
smooth muscle cells by FACS or immunohistochemistry (above 70%
identification)? 
Currently we are using for FACS analysis, a method which includes fixation
with 0.25% paraformaldehyde at 4ºc for 1 hour, permeabilization with 0.2%
Tween20 at 37ºc for 15 minutes, blocking and incubation with alpha-SMA
(clone 1A4 DAKO).

We use endothelial cells as a negative control, but there was a high
staining percentage for alpha-SMA (above 80% staining), although the isotype
controls are always stained negatively. We are not sure about the
specificity of the procedure and would like to examine it; can someone
please propose a method to prove the specificity of the staining? 

Many thanks in advance,

Yael Sarnatzki, M.Sc
Head of QC Group 

M.G.V.S  Ltd
Lady Davis Carmel Medical Center
Haifa, 34362, IL
Tel: +972-4-8250832
Fax: +972-4-8250841
Email: y.sarnatzki at mgvs.co.il
 







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