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Blood, Vol. 113, Issue 12, 2816-2825, March 19, 2009
Basophil effector function and homeostasis during helminth infection
Blood Ohnmacht and Voehringer
113: 2816
Supplemental materials for: Ohnmacht and Voehringer
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 68.9 KB)
- Figure S1. Purity of bone marrow derived basophils after depletion of mast cells (JPG, 74 KB)
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Bone marrow of 4get mice was cultured for 11 days in the presence of 0.3 ng/ml IL-3, labeled with biotinylated anti–c-Kit mAb and subjected to MACS purification (Miltenyi Biotech). Dot plots show the frequency of mast cells (IL-4/eGFP+c-Kit+), basophils (IL-4/eGFP+c-Kit−), and other cells types (IL-4/eGFP–c-Kit−) before and after purification.
- Figure S2. Expression of Thy1.2 on basophils from normal BALB/c mice (JPG, 35.4 KB)
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Splenocytes of naive BALB/c mice were stained with anti-B220, anti-IgE, and anti-Thy1.2 or isotype control. R1 indicates the basophil-gate. The dot plots on the right show the staining with anti-Thy1.2 (upper dot plot) or isotype control (lower dot plot) on gated basophils.
- Figure S3. Gating strategy to define basophil populations in the fetal liver, bone marrow, and spleen (JPG, 71.2 KB)
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E16.5 fetal liver cells were stained with anti–Ter119-PE, anti Siglec–F-PE, and anti–IL-3Ra-bio followed by streptavidin-APC. Bone marrow cells and splenocytes were stained with anti–CD4-PE-AlexaFluor700, anti–Siglec-F-PE, and anti–IL-3Ra-bio followed by streptavidin-APC. Gates define the population that was analyzed further as indicated by the arrows.
- Figure S4. Morphology of fetal liver basophils (JPG, 18.7 KB)
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E16.5 fetal liver cells were stained with anti-Ter119, anti–Siglec-F, and anti–c-Kit to gate out erythroblasts, eosinophils, and c-Kit+ cells. Cells were sorted as Ter119-Siglec-F-c-Kit-IL-4/eGFP+ cells. Pictures were acquired with an 60× oil immersion objective on a Olympus microscope BX41 equipped with a F-View II camera and cell^F software (Olympus).
- Figure S5. Detection of basophils in naïve and N. brasiliensis-infected 4get mice (JPG, 87.2 KB)
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Single cell suspensions of bone marrow (BM), spleen, peripheral blood, and lung was analyzed before (naïve) or 9 days after infection with N. brasiliensis. The basophil population can be distinguished from other cell types by their expression of IL-4/eGFP and cell surface-bound IgE (indicated region in the left panels). Only few IL-4/eGFP+ cells express c-Kit (right panel).
- Figure S6. Identification of basophils in the lung and small intestine of N. brasiliensis-infected c-KitW-sh mice (JPG, 41.5 KB)
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(A) Lung tissue was stained with anti-CD200R3 followed by Cy3-conjugated donkey anti-rat antibody. (B) Small intestine was stained with biotinylated anti-FceRI followed by amplification with TSA-biotin and detection with Cy3-conjugated streptavidin. Sections were counterstained with DAPI. Scale bars indicate 200mm.
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