Susan,

Thank you for your reply and the references.

Ray


-----Original Message-----
From: Confocal Microscopy List
[mailto:[log in to unmask]]On Behalf Of Garfield, Susan
(NCI)
Sent: Wednesday, August 23, 2000 9:57 AM
To: [log in to unmask]
Subject: Re: Upright scope with closed culture system and 100x
objective?


Hi Ray,

We are using the Bioptechs FCS2 enclosed system on an upright Nikon
microscope to look at GFP-fusion protein translocation in live cells.  We've
used both a 60x and 100x objective.  Bioptechs made a special stage adapter
for us so that we could turn over the FCS2 and image cells while perfusing
drugs through the chamber.  The chamber is also hooked up to a temperature
controller.  Here's some of the publications that used this technique:

Lorenzo, P. S., Bogi, K., Hughes, K. M., Beheshti, M., Bhattacharyya, D.,
Garfield, S. H., Pettit, G. R., and Blumberg, P. M.: Differential roles of
the tandem C1 domains of protein kinase C  in the biphasic down-regulation
induced by bryostatin 1.  Cancer Res. 59: 6137-6144, 1999.

Wang, Q.J., Bhattacharyya, D., Garfield, S., Marquez, V.E., and Blumberg,
P.: Differential localization of protein kinase C by phorbol esters and
related compounds using a fusion protein with green fluorescent protein.
Journal of Biological Chemistry. 271 (52): 37233-37239, 1999.

Wang, Q.J., Fang, T., Fenick, D., Garfield, S., Bienfait, B., Marquez, V.E.
and Blumberg, P.: The lipophilicity of phorbol esters as a critical factor
in determining the pattern of translocation of protein kinase C fused to a
green fluorescent protein. J of Biol Chem. 275 (16):  12136-12146, 2000.

Susan Garfield
Confocal Facility Manager
Laboratory of Experimental Carcinogenesis
Division of Basic Sciences
National Cancer Institute, NIH
Building 37, Room 3C28
37 Convent Drive MSC4255
Bethesda, MD  20892-4255
Phone: 301-496-5688; Fax: 301-496-0734





> -----Original Message-----
> From: ray hester [SMTP:[log in to unmask]]
> Sent: Wednesday, August 23, 2000 10:30 AM
> To:   [log in to unmask]
> Subject:      Upright scope with closed culture system and 100x objective?
>
> Hi,
>
> Is anyone using an upright fluorescence microscope to analyze viable cells
> in a closed culture system with a 100x oil objective. i.e., the cells will
> have to be upside down on the upper cover glass and the chamber filled
> with
> culture fluid?
>
> If so, did you have to make your culture holder or are there commercially
> available systems that will accomplish this?  The Bioptechs catalog shows
> the FCS3 which would permit this but I'm not sure if it's in production
> yet.
>
> Thanks for any help.
>
> Ray
>
> Raymond B. Hester, Ph.D.
> Research Cytometry Laboratory
> CSAB 357, Biotechnical Services
> University of South Alabama
> Mobile, AL 36688
> Voice:    (334) 460-6029
> FAX:      (334) 460-6073
> E-mail:   [log in to unmask]