From: PAUL HALLBERG (Paul.Hallberg@mail.tju.edu)
Date: Tue Dec 17 2002 - 11:05:47 EST
As requested, I have listed a summary of suggestions from cytometrists to
facilitate sorting CHO's. The majority of responders suggested using a
commercial product Accutase, from Innovative Cell Technologies, Inc. Others
suggested various culture techniques using a combination of trypsin and/or
EDTA. Another described a novel technology. I want to thank everyone for
their helpful tips. I will apply some of these techniques and will share
them with my clients.
Paul L. Hallberg
Flow Cytometry Manager
KCC CORE Flow Cytometry Facility
Thomas Jefferson University
Paul,
I have a few comments/suggestions. I've sorted CHOs successfully in the
past. This is what is required. (I'm going to provide all the details so
don't be offended if I mention something that is a no-brainer.)
1. Don't overgrow the cells. If the cells become confluent, they come off
in one big sheet when trypsinized and you'll have difficulty getting a
single-cell suspension.
2. Remove the medium and then add Ca/Mg-free PBS. Add just enough to
cover the cells. Gently rock the flask back and forth a little to wash
the cells then let them sit for no more than 5 minutes. Take the PBS off
and save it as some cells may have already come off. (Assuming that you
are interested in the cells that may have been lightly attached. For
instance, in the case of apoptosis assays these cells that come off easily
may be the apoptotic cells.)
3. Add the trypsin/EDTA solution. Once again, add just enough to cover
the cells. Gently rock the flask again and let it sit at RT for one
minute to two minutes. Check to see if the cells are starting to lift
with a dissection scope. If they are starting to lift, give the
flask/plate a sharp smack on the benchtop. Then look under the scope
again. This should dislodge the majority of the cells. Don't ever leave
the trypsin/EDTA solution on for more than 2 minutes. In my hands, more
than 2 minutes of trypsinization lysed some cells. That's when you really
get big clumps.
Additional/optional steps:
4. If you're having a lot of trouble getting the cells to come loose and
some cell lysis is occurring a little DNAse in the buffer can help prevent
clumping.
5. A different style plate/flask (coating?) may facilitate cell harvest.
6. If you have the capability, and it doesn't affect your experimental
design, you could culture the CHO in suspension with a rotate. (In which
case you can ignore all the previous suggestions!)
I almost forgot. Make sure you use a big enough nozzle when you do the
sorting, since the CHOs can get quite large. I think a 70uM nozzle will
be too small. 100uM is a better way to go.
David McFarland
GlaxoSmithKline
I got good results by agitating with EDTA in DMEM. Then I let the Suspended
cells sit, overnight, in RPMI w/o Phenol red. They showed up beautifully
when I stained for GP120.
Adrian Rubio M.S.
Research and Development Coordinator
Bio-Tech Imaging Inc.
2425 Ridgecrest Dr. SE
Albuquerque, NM 87108
(505) 348-8782, arubio@biotechimaging.com
Paul,
If you can use trypsin and it doesn't strip your epitope, try washing cells
with PBS and then use the smallest volume of trypsin at the lowest
concentration you can get away with. I typically use 3ml of trypsin per T75
flask. The thin film of liquid is enough to detach the CHO cells with
incubation at 37oC. Don't quench with medium containing serum but
immediately dilute trypsin with PBS and then spin down gently. Whilst I do
get some clumps, the majority are single cell suspensions.
Hope that's helpful.
Simon
Simon Rice
GlaxoSmithKline R&D UK
Hi Paul,
although we have not sorted CHO cells post detachment nor tested
VE-cadherin, we have recently had success detaching the CHO cells from
culture dishes using a fairly new product called Accutase from Pheonix
Flow Systems. The active enzyme(s) in this product are not disclosed
(see their web-site for pdf of this product) but in a preliminary
experiment we obtained a single cell suspension that exhibited very high
viability at all concentrations tested. In one parallel experiment
using the primitive hematopoietic cell line KG1a, we noted that surface
expression of CD34 (class III epitope), CD109 and CD45 (J33, pan CD45)
were not cleaved at concentrations sufficient to effect detachment of
CHO cells. Might be worth a try.
Rob Sutherland
University Health Network
Toronto
Dear Paul:
I would recommend you use single cell gel microdrop (GMD)encapsulation
technology. It prevents clumping and allows to sort single viable cells
based on the secretion of the particular protein (you will need two Abs:
biotinylated capture and fluorochrome-labeled as detection). If you are not
familiar with this technology, see our web site:www.onecell.com.
Regards,
Yevgenya Akselband, M.D., Ph.D.
Senior Scientist
One Cell Systems, Inc.
"Cell Stripper" as supplied by Cellgro (Mediatech, Inc.) is a
non-enzymatic cell dissociation solution that contains a mixture of
chelators. I've used it in the past with some epithelial cell lines and
I've noticed that some tend to clump more than others when placed in
culture...
Can't answer your question from my own experience, but you might try
keeping EDTA in your cell suspension or use mild enzyme treatment that
is not likely to affect VE-cadherin
Hi Paul:
Many of my clients have switched to Accutase which is a Phoenix Flow
product. It is a mixture of trypsin, edta,collagenase, dnase, etc specially
formulated to detach cells in a least damaging way while minimizing
clumping. It seems to work pretty well, even for Annexin V stuff on adherent
cells. Give it a try. Hope you have some happy holidays!
Joanne Lannigan, MS
Director, Flow Cytometry Core Facility
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@virginia.edu
Dear Paul,
we remove our CHO using a non-tripsin treatment (Accutase, distributed by
Innovative Cell Technologies, Inc.,
A Phoenix Flow Systems, Inc. Company, 6790 Top Gun St. #1, San Diego, CA
92121, Phone: 858.587.1716, Fax: 858.453.2117) after few washes in Ca++,
Mg++ free PBS. We use accutase to avoid removal of surface antigen during
detachment. Few minutes in the incubator and your cells are happily floating
and do not usually form clumps. We then wash the cells in PBS again and
stain them for surface markers. I don't sort them, but analyse them only, so
I cannot comment on their possible aggregation when resuspended in media,
but you could try to include in your media something like EDTA that will
chelate the Ca++ available. You'll probably have to check viability too.
Hope this help
Mara
P.S. Could you please post a summary of your responses on the list? Thanks
-Flow cytometry (in the presence of
CA++, epitope is trypsin sensitive),
use PBS + 2-5mM EDTA for cell
detachment
DATA SHEET: Mouse anti-Human VE-CADHERIN
Catalog#: RDI-VECADHabm $375.00/vial
Package Size: 100ug in 1ml (0.1mg/ml) PBS with 0.1%
BSA and 0.02% NaN3.
Species: mouse IgG2a
Clone ref: BV6
Immunogen: human endothelial cells
Reactivity: react with human VE-Cadherin.
Specificity determined by
immunoprecipitation from a detergent
extract of biotinylated cells. The
epitope recognized is located on the
extracellular domain of the
molecule. Antibody binding requires
calcium. Does not react with mouse or
bovine VE-Cadherin . Epitope is
trypsin sensitive
Uses: -western blotting (non-reducing
conditions, 2-5mMCa++ required in
buffer,expected size is approx
140kda)
-Flow cytometry (in the presence of
CA++, epitope is trypsin sensitive),
use PBS + 2-5mM EDTA for cell
detachment
-histochemistry
-immunoprecipitation
-ELISA
Titers must be optimized for each
application
Storage: Store at 4-8 DEG C
ref: -Lampugnani, G. et al (1992) J. Cell
Biol. 118:1511-1522
-Brevario, F. et al (1995)
Arterioscler. Thromb. Vasc. Biol.
15:1229-1239
-Martin-Padura, I et al. (1995) J
Pathol 175:51-57
-Matsumoto, Y et al (1993)
Transplantation 56:69-75
Precautions: For In vitro research Use Only. Not
for use in or on humans or animals or
for
diagnostics.
Note: Upon receipt, tighten cap, and centrifuge for 1-2 minutes at 1000 RPM
to
concentrate antibody in vial (during shipping, some antibody can become
lodged
inside the cap and on sides of vial.
-see other antibodies at:http://www.researchd.com/absort.htm
Sincerely,
Research Diagnostics Inc
Pleasant Hill Road
Flanders NJ 07836
phone 973-584-7093
fax 973-584-0210
email: researchd@aol.com
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