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we have an ACAS instrument too, but do not use it very frequently due to
problems with cell adhesion and difficulties getting good experimental
conditions (e.g. half confluent cell layers, stability of dye loading etc.).
Nevertheless we have done some good experiments with different cell lines.
Could you please give some more detailed information about the experiments
you want to do?
Here some information about experimental conditions used in our lab:
Preferentially we try to use adherent cells grown on plastic surfaces with
or without coating.
When this is not possible due to dim fluorescence signals or excitation by
UV light, we use cover glasses which have to be coated in most cases with
different materials depending on the cell line. So far, we had relatively
good results using cover glasses coated with e.g.:
- poly-L-lysine (20 ug/ml) for BHK21 cells (hamster),
- vitronectin or fibronectin (10 ug/ml) for M21 or other human melanoma cell
lines (poly-L-lysine may be used instead),
- in some cases coating with serum may work as well.
Sometimes it might be necessary to culture cells for more than 24 hours to
get good cell spreading and adhesion.
With respect to loading and leakage of the dyes, we found large variations.
Some cells retain loaded dyes like CFDA-AM (bright), Indo-1-AM (dim
fluorescence without Ca-influx) or Fluo-3-AM (dim witout Ca-influx)
relatively stable (10-20% loss of fluorescence within 1 to 2 hours). Some
cell lines (e.g. A431) show active secretion phenomena or loss of (FITC)
fluorescence due to acidic compartment within the cytoplasm. Some cells do
not load at all.
In kinetic experiments we have analysed loading of cells with CFDA-AM (30
min. at room temperature), washed out the dye and analysed leakage for
additional 60 to 90 min. at room temperature. In these experiments we have
sometimes observed increasing fluorescence after removal of the dye (A431,
WM 266).
Do you optimize your instrument for minimal photo bleaching prior to kinetic
experiments?
I hope this will help!
.. and, because ACAS instruments are not very "wide-spread" in the world, I
would like to hear from you and other ACAS users.
Good luck
Georg
------------------------------------------------------------------------
H.G. Kreysch
Merck KGaA
Biomed Fo/IMO - A25/410 Tel.: (49)6151 - 72 7323
Frankfurter Str. 250 FAX: (49)6151 - 72 7398
D-64271 Darmstadt E-Mail: kreysch@merck.de
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
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