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Blood, Vol. 111, Issue 5, 2755-2764, March 1, 2008
APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells
Blood Belnoue et al.
111: 2755
Supplemental materials for: Belnoue et al
Files in this Data Supplement:
- Figure S1. Similar induction of B cell proliferation by complete APRIL A88 and truncated APRIL H98 (JPG, 36.6 KB)
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Splenic B B220+ cells were purified sorted using an AutoMACS separator. 105 B cells were seeded in 96 well plates at 1 × 105 cells/well and assayed for proliferation in response to different concentrations of complete APRIL A88 or truncated APRIL H98.
- Figure S2. The main cell population in PB-supporting adult BMSCs is resident macrophages (JPG, 42.1 KB)
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(A) After 6 days of culture, enriched BMSCs from a pool of fifteen 2- or 10-week-old (adult) mice were detached with cell scrapers and stained with following antibodies: anti-Mac-1 (M1/70, Pharmingen), anti-F4/80 and anti-Gr-1 (RB6-8C5, Caltag, Birmingame). FACS histograms represent 5 independent cultures per each group. (B) Percentage of each subset expressing Mac-1, F4/80 and Gr-1 determined by FACS staining as described in panel A. Results are expressed as the mean (mean ± SD) of 5 independent cultures per age group. (C) After 6 days of culture, enriched BMSCs from a pool of fifteen adult mice were detached with cell scrapers and stained with following antibodies: MHCII (clone M5), anti-CD11c (HL-3, Pharmingen), anti-Thy-1 (clone 53-2.1, Pharmingen) and anti-VCAM (clone 429, Pharmingen). CCR2 staining was performed through its ligand, MCP-1-biotin, followed by avidin-FITC staining. FACS histograms of MHCII, CD11c, Thy-1, CCR2 and VCAM-1 staining gated on Mac-1+ cells represent 3 independent cultures.
- Figure S3. Adult enriched BMSCs do not support plasmablast proliferation (JPG, 29.5 KB)
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(A) Purified PBs (5 × 103) were added to adult BMSC in presence or absence of 0.5mM of hydroxyurea to block proliferation, or incubated in medium with/without IL-6 (controls). Surviving TT-specific IgG ASCs assessed by ELISpot after 48h, as described in Figure 1. (B) Splenic B B220+ cells were purified sorted using an AutoMACS separator. 105 B cells were seeded in 96 well plates at 1 × 105 cells/well and assayed for proliferation in response to different concentrations of LPS as described previously (Ingold et al, 2005). Hydroxyurea was added at a final concentration of 0.5mM. (C) Purified PBs (5 × 103) were labeled with 1µM of CFSE prior (t0) to culture on adult enriched BMSCs with or without addition of hydroxyurea, as described. After 24h or 48h of culture, recovered CFSE-stained PBs were pooled for each condition of culture (number of wells different for each condition i.e. different numbers of events) and CFSE dye dilution was evaluated by FACS on gated live PBs. FACS histograms represent one of three independent cultures.
- Figure S4. (A) Anti-APRIL antibody reactivity (JPG, 35.8 KB)
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5 × 104 BMSCs from adult wild-type (left) and APRIL−/− (right) mice were stained in chamber slides as described in Material and Methods. (B) FACS staining of HEK-293T cells transiently transfected with APRIL (upper) or BMSCs (lower) from adult (left) or 2wk old (right) mice with anti-APRIL antibody.
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