Further Characterization of Activin A-induced IgA Response in Murine B Lymphocytes

We have recently shown that activin A, a member of TGF-beta superfamily, stimulates mouse B cells to express IgA isotype but other isotypes. In the present study, we further characterized effects of activin A on B cell growth and IgA expression. We found that activin A did not have effect on LPS-stimulated cell viability. In parallel, CFSE staining analysis revealed that activin A did not alter cell division. An increase of IgA secretion by activin A was completely abrogated by anti-activin A Ab but not by anti-TGFbeta1 Ab. In the same conditions, no other isotypes are significantly affected by each antibody treatment. Finally, activin A, as similar to TGF-beta1, increased IgA secretion by mesenteric lymph node cells. These results suggest that activin A can specifically stimulate IgA response, independent of TGF-beta in the gut.

Fluorescent labelling of MC3T3 cell line by 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate, succinimidyl ester mixed

BACKGROUND: 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester mixed (CFSE) is the fluorescent labelling agent of living cells and used to trace the cells in vivo after transplatnation of various cells. The CFSE labelled cells can maintain fluorescence for up to 7 days after labelling. The MC3T3-E1 cell line (MC3T3) has been used for many studies about osteoblast, which is well known as a mouse preosteoblast. So the CFSE would be used to trace the transplanted MC3T3. However there are few reports about CFSE labelling of MC3T3. This study is aimed to know about adequate concenturation and incubation time of CFSE to MC3T3. MATERIALS AND METHODS: The MC3T3 was incubated in a humidified atmosphere of 95% air with 5% CO2 at 37 degrees C using alpha-minimal essential medium (alpha-MEM) containing10% FBS and gentamycin. Ten mM CFSE solution in dimethylsulphoxide (DMSO: 1%) was diluted with phosphate buffered saline (PBS) and final concentration of culture medium was, respectively, 5, 10, 15, 20, 25 and 30 microM. Then the MC3T3 was incubated with CFSE in a humidified atmosphere of 95% air with 5% CO2 at 37 degrees C for 5, 10, 15, 20, 25, 30, 35, 40 and 45 minutes in each concentration. The fluorescence of CFSE labelled cells was analysed with a inverted fluorescence microscope. The duration of cell labelling was also studied. Trypan blue dye exclusion test was done for cell viability. RESULTS: For concentration between 5 and 10 microM, CFSE did not significantly label the MC3T3 in vitro. The destruction of MC3T3 was observed at the concentration of 20 microM. In the concentration of 15 microM., the best labelling was obtained at an incubation period between 15 and 30 minutes. The MC3T3 labelled with an incubation period of 15 minutes at 15 microM. was still fluorescent 7 days after CFSE labelling. The mean cell viability was 95.93%. CONCLUSION: These results suggests an incubation period of 15 minutes at 15 microM. of CFSE provides best labelling of MC3T3 in vitro.

The research of lymphocyte proliferation using CFSE and FCM / 中国药理学通报

Aim To quantitatively analyze the lymphocyte proliferation using the time-series data of carboxyfluorescein diacetate succinimidyl ester(CFSE).Methods The model of immune suppression in mice was created by cyclophosphamide and CFSE dye was used for staining the lymphocyte of both control group and treatment group animals. The data were analyzed through mathematical model-fitting.Results The first generation of cell proliferation of the control group and treatment group were 27.17 h and 22.88 h; cell death rates in each division were respectively 20% and 40%; the half-life of cells before the proliferation was respectively 31.53 h and 43.32 h.Conclusions The mathematical fitting of CFSE data can quantitatively analyze the mechanism of the drugs attecting the proliferation of lymphocyte.In this experiment the mechanism of cyclophosphamide inhibiting lymphocyte proliferation may be due to the increase of cell death rate of each division caused by cyclophosphamide.