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Fig. S1. Ficoll treatment of donor huGPA RBCs. Anticoagulated blood obtained from huGPA mice was layered over Histopaque-1077 (Sigma-Aldrich) under sterile conditions prior to centrifugation to remove the mononuclear, according to manufacturer's instructions. The RBC-enriched pellet was then washed several times and subjected to density gradient centrifugation using Histopaque-1119 (Sigma-Aldrich) followed in order to remove granulocytes. Representative dot plots of CD45 positive leukocytes in blood from mouse huGPA (A) before and (B) after Histopaque (“Ficoll”) treatment are shown and indicate that in treated samples, these cells are undetectable. (C) Following WBC-reduction using Ficoll, huGPA RBCs were labeled with a fluorescent linker (PKH-26, Sigma-Aldrich) and transfused into WT mice. Mice were bled shortly thereafter and analyzed by flow cytometry. Representative histogram showing the PKH-negative (recipient blood) and PKH-positive (transfused/donor) RBCs are shown, and by gating on the PKH-negative or PKH-positive populations, the forward and side-scatter dot plots of PKH-negative (recipient blood, D) and (PKH-positive, E) RBC populations are indicated and appear comparable in their relative distribution. Since the transfused RBCs (PKH-positive) represent only about 5% of total RBCs, the dot plot for PKH-negative RBCs only show about 5% of the total events so that roughly equivalent numbers of cells relative to PKH-positive RBCs are displayed.

Fig. S2. Alloantibody reactivity with huGPA versus FVB RBCs. Mice (n = 12, consisting of both WT and Thal) were transfused with buffy-coat/granulocyte-depleted huGPA RBCs (Fig. S1) equivalent to 1-2 packed units with CpG-ODN adjuvant, followed by weekly transfusions of red cells alone for another 3 weeks. Since huGPA mice are on the FVB background strain, the presence of IgG-specific alloantibodies against FVB and huGPA RBCs in diluted (1 in 20) mouse plasma was measured using flow cytometry by a previously described IAT.18,19 (A) As a control, reactivity of diluted plasma from untransfused mice against RBCs from FVB and huGPA mice were tested and representative histograms show background fluorescence of those RBCs. The mean fluorescent units (MFI) are indicated for each histogram. (B) Representative histograms of FVB and huGPA RBC fluorescence showing that the transfused mouse has made a strong and specific alloantibody that is reactive with huGPA RBCs in this particular mouse. (C) Alloantibody responses against FVB and huGPA RBCs for each individual transfused mouse (#1 through #12) were expressed as MFIs and shown in the table. “Difference” is the MFI units of reactivity with huGPA RBCs subtracted from MFI units of reactivity with FVB RBCs.

Table S1. List of antibodies and clones used for multi-color flow cytometry

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