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Figure 2 | Virology Journal

Figure 2

From: Analysis of adenoviral attachment to human platelets

Figure 2

Characterization of Ad binding to human platelets. Platelets were isolated from platelet-rich plasma as described in Materials and Methods. Platelets were incubated with Ad (MOI = 10, 1 hr, RT), followed by a thorough rinse and incubation with a FITC-labeled anti-Ad hexon antibody (1:1 dilution, 4°C, 1-hr), Direct flow cytometry was used to measure Ad binding as FITC-positive platelet events (a) Platelets were gated by their characteristic forward light scatter and labeling with an anti-αIIbβ3 (α-CD41) antibody. (b) To exclude non-specific recognition of unbound platelets by the anti-Ad hexon antibody, the negative control comprised omitting Ad and incubating platelet directly with the antibody. (c) The degree of Ad attachment to platelets was measured by staining with the FITC-anti Ad hexon antibody. (d) To evaluate the effect of platelet activation on Ad binding, platelets were first activated by thrombin (0.5 U/ml, 20 min, RT), rinsed and incubated sequentially as above with Ad and stained by the anti-Ad hexon antibody. (e) To measure the effect of divalent ion supplementation on Ad attachment, MnCl2 (5 mM) was added prior to Ad incubation. (f) Enhancement of Ad attachment to platelets by sequential thrombin activation and MnCl2 supplementation. (g, h) Ad incubation activates platelets. Platelet activation was measured using annexin staining, reflecting exteriorization of phosphatidylserine, either w/o Ad (g) or w/Ad (MOI = 10, RT, 1-hr) (h). All figure data representative of at least 2 different experiments (n = 3 for each).

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