Murine model of platelet alloimmunization. (A) Experimental design to induce antiplatelet alloantibody production. BALB/C (H2^d) mice were immunized weekly for 3 weeks with 1 × 10⁸ platelets in PRP isolated from C57BL/6J (H2^b) mice. (B) IgG antibodies production in immunized BALB/C mice was evaluated 7 days after each transfusion by flow cytometry. Left panels indicate representative dot plots of the gating strategy used to identify platelets with an anti-GP1bβ antibody. Right panels indicate representative histograms of the fluorescence of Alexa-488 GaM IgG antibodies on platelets from C57BL/6J (H2^b) or BALB/C (H2^d) mice preincubated with the serum of immunized (red) or nonimmunized (blue) BALB/C mice. (C) Mean fluorescence intensity of Alexa-488 GaM IgG antibodies bound to H2^b platelets preincubated with the serum of immunized BALB/C mice (MFI ± SEM; ∗∗P < .01; 1-way ANOVA; n = 11) (D) Representative dot plots of the gating strategy used to identify erythrocytes (left upper panels) and T-lymphocytes (left lower panels). Representative histograms of the fluorescence of IgG antibodies bound to erythrocytes (Ter119⁺) (right upper panel) and T-lymphocytes (CD45⁺ CD3⁺, Ram1^-) (right lower panels) from C57BL/6J and BALB/C mice (red, immunized mice; blue, nonimmunized mice). (E) The Alloimmunized BALB/C mice were injected with 2 × 10⁸ Oregon green CFDA-SE-labeled platelets from C57BL/6J mice. Left panel indicates representative dot plots of the gating strategy used to determine the kinetics of the elimination of transfused platelets in naive or alloimmunized mice. Right panel indicates the kinetics of elimination of transfused platelets. They were evaluated by calculating the percentage of Oregon green-positive transfused platelets among 10 000 RAM.1⁺ platelets at each time point after transfusion in nonimmunized (orange) and immunized mice 1 week after the first (blue), second (green), and third alloimmunizations (red), and normalized to those obtained at time 1 minute (n = 3). FSC, forward scatter; SEM, standard error of the mean; SSC, side scatter.