Figure 7

Siglec-9 binds to csMUC16 and mediates immune cell-target cell binding. A, Siglec-9 expressing Jurkat cells were stained with CellTracker Blue and MUC16^negOVC or MUC16^posOVC cells were stained with CellTracker Green. The Jurkat cells and tumor targets were mixed in 1:1 ratio, incubated for 30 min at room temperature and conjugate formation was determined by flow cytometry as described previously [16]. Mean and standard deviation data for percent conjugation obtained from three independent flow cytometry experiments are summarized in the bar chart. B, A direct 96-well plate-based cell adhesion assay was also used to measure binding of MUC16^posOVC and Siglec-9 expressing Jurkat cells. Ovarian tumor cells were plated in 96-well plates. Calcein AM labeled Siglec-9^pos or Siglec-7^pos Jurkat cells were added to the wells. After 25 min incubation wells were washed three times and the amount of fluorescence remaining in the wells was measured. Data shown is mean of six experiments.