RESUMO
Objective To establish an accurate sorting method with less interference to the cell activity for isolating CD 3+CD4+T lymphocytes in human intestinal tissue .Methods Firstly the digestive enzymes were adopted to decompose the human intestinal tissue ,and then the cell suspension was obtained by the filtration of 40 μm cell strainer .Mononuclear cells were collected by using continuous density Percoll gradient centrifugation technique ,then the flow cytometry was adopted to sort CD3+ CD4+ T lympho-cytes in mononuclear cells .The survival rate ,cell purity and morphological observation of the sorted cells were adopted to conduct the evaluation on the sorting method .Results The digestive enzymes well decomposed human intestinal tissue into cell suspension , the purity of CD3+CD4+ T lymphocytes by using continuous density Percoll gradient centrifugation was (43 .9 ± 7 .3)% ,while the purity of CD3+CD4+ T lymphocytes was(96 .9 ± 1 .2)% after fluorescence-activated cell sorting ,the difference between them was statistically significant(P<0 .01) .The survival rate of sorted CD3+CD4+ T lymphocytes was(97 .8 ± 1 .6)% with intact cell shape . Conclusion The Percoll gradient centrifugation technique combined with the fluorescence-activated cell sorting not only collects highly pure human intestinal CD3+CD4+ T lymphocytes with little influence on the cell activity and shape ,but aslo may be carried out under sterile conditions ,the sorted cells can continue to be used in the other functional researches .