The Prefrontal Cortex : Response Selection or Maintenance Within Working Memory ?

(1987). 12. J. L. Goodman et al., J. Clin. Invest. 103, 407 (1999). IFA binding assays were performed according to the methods described. Means and medians were calculated, and the statistical significance of differences in the distribution of bacterial binding to cells between groups was tested with a Wilcoxan sum-of-ranks (Mann-Whitney) test. The Fisher exact test was used when comparing the percentage of cells infected between groups. All binding and infection experiments were repeated twice by IFA and twice by FACS (16). 13. K. L. Moore et al., J. Cell Biol. 118, 445 (1992). 14. G. S. Kansas, Blood 88, 3259 (1996). 15. mAbs used in these studies (and their sources) include the anti-CD11b (B-1 integrin subunit) clones Leu 15 (Becton Dickinson Biosciences, San Jose, CA), OKM1 and OKM10 [American Type Culture Collection (ATCC), Rockville, MD], and MO1; the antiCD15s clone CSLEX-1 (both obtained from Beckman Coulter, Fullerton, CA), the 3G8 clone directed against the Fc receptor, CD16; the ML5 anti-CD24 clone (both from Pharmingen, San Diego, CA); and the dft1 (Sigma, St. Louis, MO) and Ig10 (Pharmingen) anti-CD43 clones. Anti-CD162 (PSGL-1) mAbs used included KPL-1 (from the laboratory of G.S.K.), PL1 (Ancell, Bayport, MN), and PL2 (Beckman Coulter). Antibodies to selectin used were as follows: for P-selectin (CD62P), AC1.2 (mAb; Becton Dickinson) and polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA); for L-selectin (CD62L), clone SK11; and for E-selectin (CD62E), clone H18/7 (both from Becton Dickinson). 16. Fluorescent HGE was prepared from infected HL60 cultures incubated with the vital fluorescent dye CellTracker Green (Molecular Probes, Eugene, OR) (C. Nelson, M. Herron, J. L. Goodman, unpublished data). HGE bacteria were liberated by passage through a 25-gauge needle and washed twice in phosphatebuffered saline (PBS). Fluorescent HGE from 1 to 2 million infected HL60 was incubated for 30 min with 250,000 target cells at 22°C in 25 ml of PBS. Binding was stopped, and the cells and bacteria were fixed by addition of 0.25 ml of 1% paraformaldehyde in PBS. Cell surface binding of labeled HGE was assessed by FACS analysis of the mean cellular fluorescence of 5000 cells and expressed as the percentage of a control with no antibodies added, with subtraction of nonspecific background determined with a baseline control in which binding had been stopped by fixation immediately upon addition of HGE to target cells. This method was shown to provide results that linearly correlate with binding as determined by direct visualization by IFA. 17. F. Li et al., J. Biol. Chem. 271, 6342 (1996). 18. K. R. Snapp et al., Blood 91, 154 (1998). 19. S. Natsuka, K. Gersten, K. Zenita, R. Kannagi, J. B. Lowe, J. Biol. Chem. 269, 16789 (1994). 20. E. Svedmyr and M. Jondal, Proc. Natl. Acad. Sci. U.S.A. 72, 1622 (1975). 21. The derivation of the BJAB cells stably expressing FucT-VII is described [A. J. Wagers, L. M. Stoolman, R. Kannagi, R. Craig, G. S. Kansas, J. Immunol. 159, 1917 (1997)]. BJAB and BJAB/FucT-VII cells were each stably transfected with PSGL-1 cDNA by electroporation as described [K. R. Snapp et al., J. Cell. Biol. 142, 263 (1998)], and clones expressing comparable amounts of PSGL-1, FucT-VII, or both were generated by limiting dilution and identified by FACS analysis with the KPL-1 and HECA-452 mAbs. 22. K. E. Norgard et al., J. Biol. Chem. 268, 12764 (1993). O-sialoglycoprotein endopeptidase was obtained from Cedarlane (Homby, Ontario, Canada). 3 3 105 BJAB dual transfectant cells (stably expressing both PSGL-1 and Fuc-TVII/sLex) were preincubated in 100 ml of RPMI medium for 1 hour at 37°C either alone or with enzyme (0.06 to 0.24 mg/ml) added. The cells were then washed twice in fresh medium without enzyme, and IFA binding assays were performed as described (12). 23. A. S. Weyrich et al., J. Clin. Invest. 97, 1525 (1996). 24. K. I. -P. Jwa Hidari et al., J Biol. Chem. 272, 28750 (1997). 25. J. B. Bliska, J. E. Galan, S. Falkow, Cell 73, 903 (1993). 26. P. Cossart, P. Boquet, S. Normark, R. Rappuoli, Science 271, 315 (1996). 27. M. B. Klein, S. Hu, C. Chao, J. L. Goodman, J. Infect. Dis. , in press. 28. R. Behl et al., Thromb. Haemostasis 83, 1148 (2000). 29. M. P. Bevilacqua and R. M. Nelson, J. Clin. Invest. 91, 379 (1993). 30. This work was supported by grants from the NIH (RO1AI40952, to J.L.G.), from the American Cancer Society (RPG-96-097 CSM to G.S.K.), and from the Illinois Chapter of the American Cancer Society (9947, to K.R.S.). G.S. K. is an Established Investigator of the American Heart Association. We thank J. Ernst, J. Farber, and B. Weston for helpful comments.