Dear Charles,

I don't have specific experience with these cells. Generally speaking
for cytoplasmic staining you should incubate the cells in the dye at 37
oC . You may in fact already be doing this, you don't mention what your
loading protocol is.

*************** New Address ***************
Stephen H. Cody,
Colon Molecular and Cell Biology Laboratory,
Ludwig Institute for Cancer Research,
Post Office Royal Melbourne Hospital,
Parkville, Victoria 3055, Australia.

Tel: 61 3 9341 3150   Fax: 61 3 9341 3104
email: [log in to unmask]

> ----------
> From:         Charles Cranfield[SMTP:[log in to unmask]]
> Reply To:     [log in to unmask]
> Sent:         Wednesday, February 24, 1999 2:34 AM
> To:   [log in to unmask]
> Subject:      Calcium imaging in T cells
>
>  Hi confocal fans,
>                 this a a question for those who do, or have done,
> calcium imaging in T lymphocytes. When loading cells with an AM ester
> form of a dye (we use Fluo-3) is there any sure way to restrict the
> dye to the cytoplasm? The nucleus in lymphocytes is about 90% of the
> total cell size, and we're finding that what we're seeing using our
> confocal is mostly nuclear calcium, and not cytosolic. Does anyone
> find this a problem when trying to measure calcium fluctuations in
> these cell types? Or is it a case that nuclear calcium ion
> concentration is a good representation of cytosolic calcium
> concentration in these cells?
> Thanks in advance,
>          Charles Cranfield.
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> Charles Cranfield,
> Confocal Microscopy Laboratory,
> Swinburne University of Technology.
> Mail No. 28, PO Box 218, Hawthorn 3122,
> Victoria, Australia.
> ph +61 3 9214 8817.
> email: [log in to unmask]
> http://www.swin.edu.au/bsee/maz/webpage/webpage1.htm
>