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We wish to look at the Calcium mobilization within endothelial cells using
Indo-1. The stimulus we wish to use might be NK cells. The NK cells will be
loaded with Fluo-3 since we are also interesed in the calcium dynamics of the
stimulating cell as well. The problems with the lag between adding the cells and
then continuing to acquire data are understood however, our problem is that we
will have a nice base line for endothelial cells to which we can compare the
resulting stimulation but we will not have such a base line for the NK cells
since they will be added as the stimulus, making any data that we acquire
regarding the Fluo-3 signal relatively uninterpretable. We have thought that we
might run a baseline of the endothelial, remove the sample, continue along the
same time line, put on a sample of NK cells, run a short baseline, return the
endothelial cells to the run and then add the NK cells to them. This would give
us a short endothelial and NK baseline and then we can collect the signals and
potentially compare stimulation to baseline. Does this sound completely
foolish? Would this be accepted as valid data? Is there any other way of
accomplishing the same end result? We have considered reversing the order of
adding the cells to see what was happening in separate experiments i.e. add NK
to endothelial as one experiment and then the endothelial to the NK as a second
experiment, but we would like to do both simultaneously if we could.
Any comments and suggestions would be most appreciated.
Haywood Pyle
pyle@kfshrc.edu.sa
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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;
Web