Purification and culture methods of murine splenic B-lymphocytes
10.3969/j.issn.2095-4344.2015.02.009
- VernacularTitle:小鼠脾脏B淋巴细胞纯化培养方法的建立
- Author:
Wenjuan LI
;
Jiaqi ZOU
;
Xinxin HAN
;
Zhihui TIAN
;
Jie LIU
;
Haidong LI
- Publication Type:Journal Article
- Keywords:
Tissue Engineering;
B-Lymphocytes;
Interleukin-4;
Lipopolysaccharides
- From:
Chinese Journal of Tissue Engineering Research
2015;(2):207-212
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:B-lymphocytes are an important participant in the immunity system. Currently, magnetic beads and complement methods are mainly used to isolate and purify B-lymphocytes. However, these methods are costly or cause large cel damage and low purity, which need further improvement. OBJECTIVE: To explore the isolation and culture methods of B-lymphocytes from mouse spleen and to study suitable conditions for B-lymphocyte isolation and culture in vitro by using interleukin-4, lipopolysaccharide, CD3 monoclonal antibody or their combination. METHODS:B-lymphocytes from mouse spleen were isolated and randomly divided into seven groups, respectively treated with interleukin-4, CD3 monoclonal antibody, lipopolysaccharide, interleukin-4+CD3, interleukin-4+lipopolysaccharide, CD3+lipopolysaccharide, and no stimulation (control group). Flow cytometry was used to detect the changes in the number and proportion of T-lymphocytes, B lymphocytes, and their subpopulations under different culture conditions. RESULTS AND CONCLUSION:The number of lymphocytes peaked at 3-5 days after addition of interleukin-4. In the lipopolysaccharide group, the number of lymphocytes began to increase at 3 days, and then peaked at 5 days. T-lymphocytes disappeared after addition of CD3 monoclonal antibody, so relatively pure B-lymphocytes could be obtained after 2 days and the number of B-lymphocytes reached the peak at 3 days. The number of mature B-lymphocytes (B220+IgD+) increased significantly after addition of CD3 antibody. In al the conditions we tested, transitional B cel subset (B220+CD93+) disappeared completely after 24 hours of culture. Experimental results indicate that after addition of CD3 monoclonal antibody and interleukin-4, T-lymphocytes can be removed in mouse spleen cels cultured, but mature B-lymphocytes remain to survive and proliferate.
