Human smooth muscle cells characterization
joannelannigan at virginia.edu
Wed Nov 2 12:14:04 EST 2005
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
email: joannelannigan at virginia.edu
From: Yael Sarnatzki [mailto:y.sarnatzki at mgvs.co.il]
Sent: Monday, October 31, 2005 3:37 AM
Subject: Human smooth muscle cells characterization
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
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%
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
Lady Davis Carmel Medical Center
Haifa, 34362, IL
Email: y.sarnatzki at mgvs.co.il
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