Permeabilized cells nonspecifically bind PFN.

PI reactivity (y-axis) versus PFN reactivity (x-axis) of nonpermeabilized (left column of panels) and NP-40 permeabilized (right) Jurkat cells. PFN untreated cells that were exposed to isotype control mAb or anti-PFN mAb are shown respectively as panels A-B and C-D. Similarly manipulated subsets were treated with PFN containing YT granule extract for 1 hour (37°C) and stained with either control antibody (E-F) or anti-PFN antibody (G-N). The concentrations of PFN were 300 ng/mL (E-H), 150 ng/mL (I-J), 75 ng/mL (K-L), and 37.5 ng/mL (M-N). These concentrations were estimated based on densitometric calculations where granules from 10⁶ YT cells yield approximately 3 ng of PFN. The resultant dot plots have intensity of PI reactivity on the y-axis (FL3) and PFN reactivity on the x-axis (FL1). Gates were established based on the antibody control groups (A-D) allowing the quadrants to be defined as follows: upper left, PI⁺ PFN⁻; upper right, PI⁺PFN⁺; lower left, PI⁻PFN⁻; and lower right, PI⁻ PFN⁺. The percentage of events in each quadrant is provided.