Ct-CD45 molecules are released into the supernatant upon stimulation of phagocytes with zymosan and PMA. (A) Monocytes stimulated with PMA for different time periods were stained with annexin-V and PI. Percentages of positive cells are indicated. (B) ct-CD45 was isolated from cell culture supernatants of monocytes stimulated with PMA or zymosan by immunoprecipitation with mAb 8-301. As a positive control recombinant ct-CD45 diluted in the same amount of RPMI was precipitated, as a negative control precipitation was performed from RPMI (medium ctrl). Isolated ct-CD45 was immunoblotted and detected by mAb 8-301. As a loading control, the light chain of mAb 8-301 used for precipitation, detected with sheep-anti–mouse-HRP, was used. Data represent at least 2 independently performed experiments. (C) Release of ct-CD45 was accompanied by the release of LDH. The experiment shows the LDH values of the supernatants used for immunoprecipitation in (B). (D) The phosphatase activity of released ct-CD45 was assessed by precipitation of ct-CD45 with mAb 8-301 from supernatants of granulocytes (7 × 107 cells) incubated with or without PMA for 4 hours. The figure shows mean values of relative fluorescent units from 4 different donors of 2 different experiments (± SD). A paired Student t test was performed (**P < .05).
