A novel method of microwave treatment for detection of cytoplasmic and nuclear antigens by flow cytometry.
Flow cytometry has recently become a useful technique for the quantitative analysis of cytoplasmic and nuclear antigens. We report here a rapid, simple, reproducible, and sensitive method for the simultaneous detection of cytoplasmic and nuclear antigens by flow cytometry. This technique involves the treatment of cell suspensions with 60 s of microwave oven heating after fixation with 2% paraformaldehyde. Following this treatment a number of cytoplasmic and nuclear antigens were detected on the human myelomonocytic cell line U937 (CD68, PCNA and Ki-67), peripheral blood leukocytes from both normal donors and leukemia patients (CD68, lipocortin-1 and PCNA) and a rat mesangial cell line 1097 (desmin, alpha-smooth muscle actin) using a standard indirect immunofluorescent staining with mouse monoclonal antibodies (mAbs). There are several advantages of this technique over the routinely used methods currently available. Firstly, microwave treatment is a rapid, simple, and reproducible method, which largely reduces both time and cost expenditure, and makes this technique widely available for flow cytometric analysis in many areas of diagnostic and research purposes. Secondly, microwave treatment produces optimal results for simultaneous detection of both cytoplasmic (CD68, lipocortin-1, desmin, alpha-smooth actin) and nuclear (PCNA, Ki67) antigens. Thirdly, microwave treatment also produces a discrete profile for DNA content analysis. Finally, microwaving retains a clear discrimination between cells and debris as measured by light scatter. This study demonstrates that microwave treatment is a powerful technique which will be particularly applicable to flow cytometric analysis in the detection of many cytoplasmic and nuclear antigens.
Lan
H Y
HY
Department of Nephrology, Monash Medical Centre, Vic, Australia.
Hutchinson
P
P
Tesch
G H
GH
Mu
W
W
Atkins
R C
RC
D000818:Animals; D017305:Annexin A1 / Q000032:analysis; D000941:Antigens / Q000032:analysis*; D015703:Antigens, CD / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry*; D003893:Desmin / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000032:analysis; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D008872:Microwaves; D009687:Nuclear Proteins / Q000032:analysis*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D051381:Rats; D000097382:CD68 Molecule
D017305:Annexin A1; D000941:Antigens; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D003893:Desmin; D015395:Histocompatibility Antigens Class I; D017493:Leukocyte Common Antigens; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C067980:CD68 antigen, human; D000097382:CD68 Molecule