Julie:
I spoke with a friend of mine from Abbott Diagnostics Division who helped
develop the Abbott ICA kit:  He volunteered the following:

The basic problem with MCF-7 is that the cell genetic population is
heterogenous. Its a mix of positive and negative ER cells. The genetic
negatives tend to overgrow the positives with time in culture and apparently
are more stable in the frozen state. Once you gain positivity with the cell
line, through selective culturing, as we did here at Abbott, you must freeze
it down to ensure retaining that activity for future use. Our line has lost a
lot of its activity. To recapture it ,we would have do a cloning process to
isolate and grow up the few stong producers left, but, since we no longer
manufacture this product, there are no plans to do this.   ATCC cell lines are
not known for their ER activity. They are stored in DMSO, which is a problem
for these cells, and does make them look sickly on slides, as opposed to other
storage medias. I'm not surprised that they have poor activity for ER.
Trypsin and physical stripping of the cells from culture flasks has no effect
on the cells or the ER activity. ER activity with MCF-7 is not , unfortunatly,
always a given. I suspect that its the line itself that does not have good ER
activity.
Jackie O'Connor



                                                                                                              
                    "Julie Clark"                                                                             
                                                     
                    edu>                 cc:                                                                  
                                         Subject:     Re: MCF-7 cells                                         
                    08/08/2002                                                                                
                    11:08 AM                                                                                  
                                                                                                              
                                                                                                              




Thank you Jackie,

I started here in November.  Shortly before I started, they had lost their ER
positivity in the MCF-7 cells which they had used for years as controls.  I
know very little about cell culture.  I suspect a few things.  A girl who
worked here during that time was not the best at sterile technique.  They were
storing the cells in the -80 freezer instead of liquid nitrogen for long
periods of time.  But, the times they did culture them right after arrival,
they still were not getting them to be positive.  Then, once they made the
slides, they were storing them in a -20 freezer that has a defrost cycle.  I
tested for mycoplasma since the cells were kind of sick looking and it was
negative.  They started using anti-mycotic shortly after that, which we have
since stopped using.  We are using pen-strep.

I guess my questions are as follows:
*What exact media formula did you use?
*We are using cell stripper for splitting and harvesting.  Is this damaging
the cells to the point we can't get any positivity.  Is trypsin worse, or
would just scrapping them during harvesting be even worse.  Does trypsin
really have an effect on cell membrane markers like EGFr?  As far as I know,
they were using cell stripper for a long period of time prior to the cells
losing their ER.
*What was your method for splitting and harvesting?
*What confluency is best for the greatest ER positivity?

We get the cells from ATCC.  If we can't start them that day, they are in
liquid nitrogen briefly.  We use the media formulation they recommend.
After we harvest the cells, they are placed in Modified Cytolyte (if you're
not familiar with the ThinPrep, it is their fixative with 1% buffered formalin
added to it) overnight on their side in a 15ml falcon tube.  They are then
spun down at 2000 for 10 minutes, 3 times.  We then use varying concentrations
of stock to determine the correct amount to add to the PreservCyte vial (Cytyc
ThinPrep again) to make our slides.  The slides come out of 95% alcohol, are
tri-fixed in acetone-methanol-formalin, rinsed and stored at -20 in freezing
media (the gross glycerol stuff we use to freeze the frozens in for the old
Abbot ER-ICA kits).  The cells have to be fixed in the modified cytolyte
because our patient samples are.  The reason they are fixed that long is
because we get samples in FedEx from other sites, so we try to leave them all
in the fixative the same amount of time.  We then toss the cells when we have
enough control slides instead of trying to maintain them until we run out of
slides.
Sorry for the lengthy e-mail.  I just thought if you knew what we were doing,
you could give us a better answer.

Julie Clark
>>>  08/08/02 10:34AM >>>

Abbott used to sell MCF-7 cultured cells as ER controls.  I used to support
that product line until they discontinued it in 2000.  At that time it was
only being sold outside the US.   I'm sure I can answer any and all of your
questions - please contact me directly.

Jackie O'Connor
Abbott Laboratories
GPRD
Discovery Cancer Chemotheraputics
Jackie.O'Connor@abbott.com



                    Julie Clark

                                     cc:

                                         Subject:     MCF-7 cells

                    08/08/2002

                    10:13 AM







Has anyone used MCF-7 cultured cells (that you have grown yourself) for ER
positive controls?

If so, can you contact me please, I have some questions.

Thanks!

Julie Clark
Technical Director
Breast Cancer Prevention Center Research Laboratory
Room 3021 Lied
Univ. of Kansas Med. Center
Kansas City, KS
Phone: Office: 913-588-3963
            Lab:     913-588-3917
FAX: 913-588-3821
Page: 913-917-6052