Flow cytometric analysis of specific binding of biotinylated CCL3 and CCL4 to L540 cells.

1 × 10⁶ cells resting (1) or stimulated for 72 hours with plastic-bound anti-CD30 agonistic mAbs M67 (10 μg/mL) (2) were incubated with biotinylated CCL3 or CCL4 and stained with avidin-FITC reagent. Controls for specificity of binding were performed using non-biotinylated CCL3 and CCL4. Compared with resting cells (1), stimulation with M67 (2), but not with nonagonistic anti-CD30 mAb BerH2, induced a clear-cut increase in both CCL3 and CCL4 binding. Stimulation with 15 ng/mL PMA + 500 ng/mL ionomycin did not modify the binding of CCL3 and CCL4 (not shown), compared with basal conditions (1). The experiment depicted is representative of 5.