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Below I've summarized our tests of using high-molecular weight dextran
to keep cells in suspension. We did this to prevent settling of cells
during up to 10 minutes of continuous acquisition for 'live' measurements
of antibody binding rates in an unstirred FACScan sample tube. I have no
experience SORTING with the dextran.
[This is a reposting of a message sent to the cytometry list in 7/93.]
We have done some trials using Pharmacia Dextran T-500 (half million av. kD)
as a viscosity-increasing agent to minimize settling of lymphoyctes during
continuous FACScan analysis over 10 min for kinetic binding purposes. I've
used this previously to keep killer and target cells from falling down into
contact with each other (J. Immunol. 115:261, 1975). Microscopic
observation (2X obj. thru the bottom of a test tube) indicated that the
lowest concentration which largely prevented sedimentation during a 20 min
period was 5% w/v. Ostwald viscometry indicated the following viscosities
relative to water: 10%, 17-18; 5%, 6; 2.5%, 2.8.
We then did pilot FACScan work with 4% dextran. It reduced the event rate
2.9-fold (a bit less than expected from the viscosities). During 7 min of
unstirred continuous acquisition, the live event rate displayed on the
screen decreased only 3%, whereas in the absence of dextran, the rate
dropped 15% in 2 min and 75% in 10 min. These figures are for a total
volume of 0.25 ml/tube.
The dextran affected the binding of a FITC-conjugated anti-LFA-1
antibody. Half-maximum binding occurred twice as fast; we gather
that this may be an effect of the dextran tying up water
molecules, increasing the effective concentration of the antibody
in the remaining free solvent (but I've not seen any authoritative
discussion of this -- I'd like to know where to look).
Surprisingly, the maximum FL1 intensity decreased about two-fold.
Possibilities which occur to us are an effect of the dextran on
the quantum efficiency of the FITC, or an effect on LFA-1
epitopes. We have not pursued this.
The bottom line is that 4% dextran reduces sedimentation in a
quarter-ml sample sufficiently to keep the rate constant for >7
min.
Technical tip: High molecular weight dextran is hard to dissolve.
Vortex at top speed IMMEDIATELY upon adding it to aqueous medium
to prevent forming an intractable lump. Vortex long and hard.
Centrifuge to get rid of the foam. We use 50 or 250 ml screw-cap
centrifuge tubes.
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Eric Martz, Professor of Immunology emartz@microbio.umass.edu
Dept Microbiology Voice: 413-545-2325 FAX: 413-545-1578
Morrill IVN 203, Box 35720, Univ Massachusetts, Amherst MA 01003-5720
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