Cytotoxicity Assay

Abstract

STARFISH inhibitor was dissolved in double-distilled, deionized water. Stock solutions of puri®ed SLT-I and SLT-II were prepared at concentrations of 400 ng ml -1 and 2 mg ml -1 , respectively, in unsupplemented MEM. Serial dilutions, in unsupplemented MEM, of inhibitor solution were prepared using a 96-well microtitre plate. Next, 5 ml of stock SLT-I or SLT-II solution was added to each well (to 80 ml ®nal volume) of the appropriate rows in the dilution plate. The solution in each of the dilution plate wells was thoroughly mixed and the microtitre plate was incubated for 1 h at 37 8C, after which 20 ml from each well was transferred to the corresponding well of a 96-well microtitre plate containing con¯uent Vero cell monolayers and 200 ml of MEM supplemented with fetal bovine serum. The Vero cell microtitre plate was incubated for an additional 48 h in a 37 8C incubator in an atmosphere of 5% CO 2 /95% air. The Vero cell monolayers were then ®xed with methanol and cytotoxicity was measured as described 11 . Crystallography A solution of the complex was made by adding 15 ml of a solution of STARFISH (0.35 mM in water) slowly to 15 ml of SLT-I B subunit (10 mg ml -1 , 0.1 M NaCl and 10 mM Tris pH 8.0) with agitation. We mixed this solution with an equal volume of reservoir solution (28% saturated ammonium sulfate, 2% 2-methyl-2,4-pentanediol, 0.1 M NaCl and 0.1 M Hepes pH 7.0) for hanging drops. Crystals grew at room temperature as clusters of needles. Diffraction data, collected on a MAR345 detector mounted on a Rigaku rotating anode generator, extended to 2.23 A Ê resolution (Table 1). A molecular replacement solution was obtained in AMoRe 24 using the B subunit of the crystal structure of the SLT-I B-subunit pentamer complexed with Gb 3 (ref. 6) as a model. 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