The long pentraxin PTX3 specifically binds to Jurkat cells after apoptosis induction.

Jurkat leukemia cells, either untreated (A) or after CD95 cross-linking (6 hours) (B), were incubated with biotinylated PTX3 (50 μg/mL). Bound PTX3 was revealed by addition of FITC-streptavidin (solid profiles). The background fluorescence in the absence of biotinylated PTX3 is also reported (light profiles). (C) Biotinylated PTX3 was separated on a 5% to 10% native PAGE and stained with silver nitrate. Lanes are as follows: (1) biotinylated PTX3, (2) conditioned medium from cells transfected with PTX3, (3) purified PTX3. Molecular weight markers are indicated on the left. (D) Jurkat cells undergoing CD95-triggered apoptosis were treated with increasing concentrations (x-axis) of biotinylated PTX3, and the extent of the binding was assessed by flow cytometry after addition of FITC-streptavidin. Results are expressed as relative fluorescence intensity (RFI) (y-axis), calculated by dividing the mean fluorescence intensity in the presence of PTX3 and FITC-streptavidin with that obtained in the presence of FITC-streptavidin alone. (E) Jurkat cells were preincubated with unlabeled PTX3, CRP, SAP, or BSA (500 μg/mL) before addition of biotinylated PTX3 and FITC-streptavidin and analysis by flow cytometry. Results are expressed as inhibition percentage, calculated as described in “Materials and methods.” (F) Binding of PTX3 (50 μg/mL) and FITC–annexin V (5 μg/mL) to apoptotic Jurkat cells was performed in the presence or absence of divalent cations. Results are expressed as mean fluorescence intensity (MFI).