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Blood, Vol. 113, Issue 23, 5878-5886, June 4, 2009
Impaired B-cell development at the pre-BII-cell stage in galectin-1–deficient mice due to inefficient pre-BII/stromal cell interactions
Blood Espeli et al.
113: 5878
Supplemental materials for: Espeli et al
Files in this Data Supplement:
- Table S1. Oligonucleotides used for production of the DNA constructs (PDF, 41.5 KB)
- Table S2. Antibodies and reagents used for flow cytometry and confocal microscopy (PDF, 63.8 KB)
- Table S3. Percentage of cells with a relocalized pre-BCR in pre-BII/OP9 co-cultures and in presence of various inhibitors (PDF, 56.6 KB)
- Figure S1. Cell sorting procedures of BM B-cell sub-populations (JPG, 271 KB)
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(A) sorting of small and large pre-BII cells. Mouse BM cells enriched for B cells were stained with antibodies specific for CD19, Igκ, Igλ and CD117. Pre-BII cells were sorted by gating out immature B cells on the basis of Igκ and Igλ expression and pro-B/pre-BI cells on the basis of CD117 expression. Large and small pre-BII cells were separated on size criterion using the Forward scatter (FSC) parameter. CD2 expression by small and large pre-BII cells is shown. (B) sorting of pro-B/pre-BI cells. Staining was performed as in section A, except that B220 replaces CD19. After gating out immature B cells (B220+Igκ/λ+) pro-B/pre-BI cells were sorted on the basis of CD117 expression. (C) purification of pro-B/pre-BI cells amplified in vitro. After in vitro expansion of pro-B/pre-BI cells by co-culture with OP9 cells in the presence of IL-7, CD2+ cells that passed the pre-BCR checkpoint were gated out. Live cells were selected based on 7-AAD exclusion.
- Figure S2. Integrin expression by large pre-BII and OP9 cells (JPG, 84.5 KB)
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Sorted large pre-BII cells and OP9 cells were analyzed by flow cytometry using anti-α4, -β1, -β7, -VLA-5 (α5β1), and -αL integrins mAbs. Isotype controls are shown (dotted line).
- Figure S3. Purity of recombinant soluble proteins (JPG, 94.6 KB)
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Purified recombinant soluble proteins (10µg) were loaded on SDS-PAGE (15% acrylamide). After migration, proteins were revealed by Coomassie Blue coloration. Molecular weights are indicated.
- Figure S4. Pro-B/pre–BI-cell differentiation in the absence of the IL-7 amplification step (JPG, 218 KB)
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(A) Sorted bone marrow pro-B/pre-BI cells (B220+ CD117+) were allowed to differentiate in the same conditions as described in Fig. 5, but without the initial step of IL7 cell amplification. Cell differentiation was evaluated by flow cytometry by analyzing CD2 expression. Results are representative of 2 experiments. (B) Sorted CD2− large pre-BII cells were incubated 1h at 37°C with OP9-pSR cells in presence of mSLCδδ, with OP9-shG1 cells in presence of mSLC, or without OP9 stromal cells. The percentage of phosphorylated BLNK cells was analyzed by flow cytometry using an anti-pBLNK mAb and compared to its isotype control (left panel). FSC = Forward Side Scatter.
- Figure S5. De novo pre–BII-cell differentiation is impaired in sub-lethally irradiated GAL1−∕− mice (JPG, 60.9 KB)
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C57Bl/6 (n=3) and GAL1 deficient (n=3) mice were sub-lethally irradiated (5 Gy) and de novo BM B-cell differentiation was analyzed by flow cytometry after 10 days, as described in Fig. 6. The absolute numbers ± SD of pro-B/pre-BI cells (CD19+IgM−CD25−), pre-BII (CD19+IgM−CD25+) and immature B cells (CD19+IgM+) are shown in the diagram.
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