Isolation of intestinal mononuclear cells from colonoscopic biopsies for immunofluorescence analysis by flow cytometry.
A method for isolating and characterizing intestinal lymphoid cells from colonoscopic biopsies is presented. Intraepithelial lymphocytes were separated from the lamina propria by incubation in edetic acid (EDTA) and lamina propria lymphoid cells isolated by incubation in collagenase followed by Ficoll-Hypaque density flotation. Quantitation of T lymphocyte helper (OKT4) and suppressor (OKT8) cells was performed using monoclonal antibodies to cell surface markers and analyzed on a flow cytometer. The isolation procedure yielded approximately 400,000 lamina propria cells and 100,000 intraepithelial cells per sample, with better than 90% viability. Surface marker analysis demonstrated significant differences in the ratios of helper to suppressor cells between the intraepithelial lymphocytes and the lamina propria lymphocytes. These demonstrate the feasibility of lymphoid cell isolation from colonoscopic biopsy specimens for surface marker analysis by flow cytofluorimetry. These techniques could prove important in the study of immune mechanisms in inflammatory bowel diseases.
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis; D001706:Biopsy; D003106:Colon / Q000276:immunology / Q000473:pathology*; D003113:Colonoscopy; D004492:Edetic Acid; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008214:Lymphocytes / Q000145:classification*; D013601:T-Lymphocytes / Q000145:classification; D006377:T-Lymphocytes, Helper-Inducer / Q000145:classification; D050378:T-Lymphocytes, Regulatory / Q000145:classification
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D004492:Edetic Acid