Fig. 9. FACS analysis of bio-PG binding to colonic lymphocytes isolated from gastrin knockout mice.

Single cell suspension was prepared from colons of two Gas-KO mice as described in Material and Methods. Experimental sample (10⁶ cells) was successively incubated with bio-PG (100nM), str-PE (1:100) and CD45-FITC (0.2μg) in 100μl binding buffer for 30-60 minutes at room temperature in the dark. Control sample was stained in the same way but incubation with bio-PG was omitted. Stained samples were washed, resuspended in DAPI containing buffer (PBS) and processed by flow cytometry. Dead (DAPI stained) cells (~30%) were excluded from this analysis by appropriate gating (not shown). (Top) Representative forward/side (light) scattering characteristics of dissociated colonic cells in control sample. Similar data were obtained for experimental sample (not shown). The oval gate shows the lymphocyte-containing cell fraction which was analyzed in the bottom panels. (Bottom) The lymphocyte fractions for the control (left) or experimental (right) sample, were concomitantly analyzed for CD45 expression (FITC fluorescence; ordinate) and bioPG/str-PE binding (PE fluorescence; abscissa). The numbers in respective gates indicate a proportion of PE-positive cells in the CD45⁺ or CD45^- subpopulations. No significant binding of bio-PG/Str-PE to CD45⁺ cells was detected (0.08% on the right experimental panel versus 0.07% on left control panel).