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>Sum LAM

You have described a problem that has been shared with us here at
Bioptechs numerous times.
We have developed a solution for this problem.  We plate our cells on
the basal surface of the transwell insert in the following manner.

1. A bio-compatible piece of tubing is cut into a small cylinder of
suitable geometry as to provide a shallow well and fluid barrier when
placed over the distal end of the insert.
2. A plug made of silicon is placed into the tubular portion of the
insert to prevent leakage through the membrane.
3. The insert is inverted (membrane side up) and cells are poured
into the well defined by the cylindrical tubing surrounding over the
membrane.
4. The cells are returned to the incubator and allowed to plate in
this inverted orientation for 45 minutes.
5. The plug and tubing are removed from the insert and the insert is
returned to the tray with appropriate media and allowed to divide
until confluent.  We have had much success with this plating
technique.

6. When the cells are confluent the insert is placed into a Bioptechs
Delta T4 Transwell Adapter which is then lowered into a Delta T dish
on the microscope.   The cells are now facing down for easy imaging
through the coverglass bottomed self-heating Delta T dish.  See
Bioptechs web site for details on equipment and contact Bioptechs
directly for additional information regarding membrane insert
micro-observation accessories.
7. Cells on the membrane can be perfused on either the apical or
basal surface during microscopy if necessary.

Dan




>Dear all,
>
>I have tried to grow cells on transwells and
>coverslips and observe tight junctions of the cells in
>FITC / TRITC channels under confocal. It was found
>that the signal is blurrer from transwells, i.e., a
>sharp spot from coverslips (about 2-3um) and a bigger
>and blurrer spot from transwell (about 8-9um).
>It was suggested that the refraction of laser on the
>transwell. Could it be a reason?
>Would anybody suggest any other reason for the
>blurring effect on the transwell?
>
>Thanks a lot!
>
>Sum LAM
>


--
Dan Focht
Bioptechs
3560 Beck Road
Butler, PA 16002
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www.bioptechs.com
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