Mouse PLT preparation and erythrocyte and leukocyte depletion

PLTs were isolated 4 days after the last DT injection (on day 8). Mice were anesthetized with an i.p. injection of ketamine (100 mg/kg) and xylazine (20 mg/kg) and blood was collected from the aorta into acid citrate dextrose anticoagulant. The entire preparation of washed PLTs was performed at room temperature. Mouse blood was centrifuged at 2300g to obtain PLT rich plasma. The PLT count was determined using a Scil Vet abc plus hematology analyzer to adjust the subsequent washes to a concentration of 600,000 PLTs/μL. After incubation for 10 min, PLT rich plasma was centrifuged at 2200g and the PLT pellet was resuspended in Tyrode’s albumin buffer (5 mM Hepes pH 7.35, 0.35% human serum albumin) supplemented with 0.5 μM PGI₂ and 10 U/mL heparin. After incubation for 10 min, 0.5 μL/mL PGI₂ (10⁻³ M) was added and the PLTs were centrifuged at 1900g. This washing step was performed a second time and the PLTs were finally resuspended at 300,000/μL in Tyrode’s albumin buffer containing 0.02 U/mL apyrase. These PLT preparations were used for immunofluorescence and in vitro translation experiments.

For RNA analyses, blood samples were first centrifuged on Histopaque 1077 (Sigma-Aldrich) supplemented with 0.5 μM PGI₂ (10⁻³ M) and 10 U/mL heparin at 250g for 30 min, after which washed PLTs were prepared from the interface containing PLTs. Residual numbers of erythrocytes and leukocytes were counted on a Scil Vet abc plus analyzer. The preparations were then depleted of erythrocytes and leukocytes by incubation with Dynabeads coated with the monoclonal antibodies Ter119 and 30-F-11 (anti-mouse CD45) (both from Biolegend; 1 μg antibody/25 μL beads, as recommended), using a ratio of 3 antibody-coated beads per target cell and two serial depletion steps. Depletion was controlled by FC analysis of 10⁶ PLTs (S5 Fig) and the ratio of residual leukocytes to PLTs was always less than 2/10⁶.

For transfusion experiments, washed reticulated PLTs were prepared from three DT-treated mice having PLT counts between 200 and 400 10³/μL. The washed PLTs were resuspended at 1.2 10⁶/μL and aliquots of the suspension were injected retro-orbitally into mice expressing tdTomato or EGFP protein (150 μL/mouse). Blood samples were collected 15 min after transfusion (time 0) and then 1, 3, 6, 9, 24, 48 and 72 h later.

In all ex vivo incubations, washed PLTs (in Tyrode’s albumin buffer) were finally resuspended at 300,000/μL in Tyrode’s albumin buffer mixed with DMEM (1/1, v/v) supplemented with 0.02 U/mL apyrase.