Journal:

Article Title: Cultured basophils but not cultured mast cells induce human IgE synthesis in B cells after immunologic stimulation

doi: 10.1046/j.1365-2249.1998.00474.x

Figure Lengend Snippet: Production of IgE, IgG4 and soluble CD23 by normal B cells mixed with cultured human mast cells or basophils. Mast cells or basophils were stimulated for 2h with antigen after sensitization with murine IgE, washed, added to highly purified B cells, and then cultured for 12 days. Stimulated mast cells were incubated with B cells in the absence or presence of recombinant IL-4. Stimulated basophils were incubated with B cells in the absence or presence of two neutralizing anti-IL-4 and anti-IL-13 MoAbs or a soluble form of CD40. Supernatants were collected and measured for concentrations of IgE, IgG4 and soluble CD23. The net synthesis of IgE and IgG4 was determined, as described in Materials and Methods. Results represent the mean ± s.e.m. of triplicate cultures. One of two comparable experiments is shown. IgE production by B cells was neither induced by unstimulated basophils, nor significantly affected in the cultures containing B cells plus stimulated basophils by the addition of control antibody or IgM (data not shown).

Article Snippet: Reagents, antigen, and antibodies Recombinant human stem cell factor (SCF) and IL-6 were generous gifts from Kirin Brewery Co. (Maebashi, Japan).

Techniques: Cell Culture, Purification, Incubation, Recombinant, Control

Journal:

Article Title: Cultured basophils but not cultured mast cells induce human IgE synthesis in B cells after immunologic stimulation

doi: 10.1046/j.1365-2249.1998.00474.x

Figure Lengend Snippet: Analysis of germ-line and mature Cε mRNA expression in normal B cells mixed with cultured human mast cells or basophils. The cell pellets obtained in Fig. 5 were used for the analysis of germ-line and mature Cε transcription. Highly purified B cells were incubated with medium alone (lane 1) or with stimulated mast cells in the absence (lane 2) or presence (lane 3) of recombinant IL-4. Stimulated basophils were incubated with B cells in the absence (lane 4) or presence of two neutralizing anti-IL-4 and anti-IL-3 MoAbs (lane 5) or a soluble form of CD40 (lane 6). After extraction of total cellular RNA, mRNA for germ-line Cε, mature Cε, and G3PDH was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), subjected to electrophoresis, and then visualized by ethidium bromide. One of two comparable experiments is shown. Neither germ-line nor mature transcripts were detected in the cultures containing B cells plus unstimulated basophils (data not shown).

Article Snippet: Reagents, antigen, and antibodies Recombinant human stem cell factor (SCF) and IL-6 were generous gifts from Kirin Brewery Co. (Maebashi, Japan).

Techniques: Expressing, Cell Culture, Purification, Incubation, Recombinant, Extraction, Amplification, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Electrophoresis

Journal: Cell reports

Article Title: Osteocyte intrinsic TGFβ signaling regulates bone quality through perilacunar/canalicular remodeling

doi: 10.1016/j.celrep.2017.10.115

Figure Lengend Snippet: (A–D) qPCR analysis of PLR genes Mmp13, Mmp14 and Ctsk and Serpine1 upon TGFβ (5ng/mL) treatment in MLO-Y4 (A, B) and OCY454 (C, D) cells. (n=3 replicates/group). (E, F) Intracellular pH (pHi) of MLO-Y4 cells after 3 days of TGFβ (5ng/ml), TβRI inhibitor SB-431542 (10 μM), or recombinant sclerostin (rhSCL, 10 ng/ml). The representative image (E) shows the shift in the emission peak from 580 nm to 640 nm after TGFβ treatment of MLO-Y4 cells. Scale bar, 100 μm). TGFβ-induced acidification is blocked by SB-431542 (F) (n=4 replicates/group). Error bars indicate mean ± SD of 3 independent experiments, *p<0.05 different from control mRNA, a-p<0.05 different from control pHi, b-p<0.05 different from TGFβ pHi, and c-p<0.05 different from rhSCL pHi. Statistics calculated from Student’s t test.

Article Snippet: For treatment, cells were cultured in α-MEM containing 0.5–1% fetal bovine serum, supplemented with 5 ng/ml TGFβ1 (Humanzyme, HZ-1011), 10 μM SB431542 (Sigma, S4317) or 10 ng/ml recombinant human sclerostin (rhSCL, R&D Systems) for the indicated times.

Techniques: Recombinant, Control

Journal: Cell reports

Article Title: Osteocyte intrinsic TGFβ signaling regulates bone quality through perilacunar/canalicular remodeling

doi: 10.1016/j.celrep.2017.10.115

Figure Lengend Snippet: (A, B) TβRII-stained osteocytes (A) (arrow, scale bar, 50 μm) in the femoral cortical bone from WT and TβRIIocy−/− mice (8-week old males) were quantified as percentage of positively stained osteocytes normalized to total bone area (B) (n=5 mice/group) (C) qPCR analysis of TβRII and Serpine1 in WT and TβRIIocy−/− femoral bones. (n=8–10 mice/group). (D, E) Silver nitrate stained images of WT and TβRIIocy−/− femoral cortical bone shows the osteocyte lacuno-canalicular network (D) and canalicular length (E) (scale bar, 20 μm, n=5 mice/group). (F, G) qPCR analysis of PLR genes, Mmp2, Mmp13, Mmp14, Ctsk, and Acp5 (F) and OCY-specific genes, Sost, Dmp1 and Phex (G) in the WT and TβRIIocy−/− bones (n=8–10 mice/group) (H, I) IHC of MMP13, MMP14, CTSK and H&E staining of WT and TβRIIocy−/− femoral cortical bone. Arrows in the image indicate positively stained osteocytes (H) that were quantified and normalized to total bone area (I), (n=4 mice/group).(J–M) SRμT shows volume (J), degree of anisotropy (K), orientation (L) and mineralization (N) of osteocyte lacunae of WT and TβRIIocy−/− bone (n=3–4 mice/group). Error bars indicate mean ± SEM with *p<0.05 compared to WT from Student’s t test.

Article Snippet: For treatment, cells were cultured in α-MEM containing 0.5–1% fetal bovine serum, supplemented with 5 ng/ml TGFβ1 (Humanzyme, HZ-1011), 10 μM SB431542 (Sigma, S4317) or 10 ng/ml recombinant human sclerostin (rhSCL, R&D Systems) for the indicated times.

Techniques: Staining