>Further, sample settling is, of course, a problem and the informal
>documentation recommends against using sample agitate while sorting,
>but suggests the addition xanthan gum at 0.1% concentration. Any
>experience with this vis a vis, fluidics effects (viscosity of the
>sample stream will go up), sorting efficacy, sterility of sorted
>cells?
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 emartz@microbio.umass.edu
Professor of Immunology Voice: 413-545-2325 FAX: 413-545-1578
Morrill IVN 203, University of Massachusetts, Amherst MA 01003
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