Expression of functional antigens on neutrophils. Effects of preparation.
We have studied how different conditions of cell labelling and isolation affect the expression of five functional antigens on neutrophils from healthy subjects. Fluorescein isothiocyanate conjugated (FITC) antisera specific for the C3bi receptor CR3 (CD11b), aminopeptidase N (CD13), the LPS:LPS binding protein receptor (CD14) and the receptors for human IgG (Fc gamma RII CDw32 and Fc gamma RIII CD16) were incubated with (i) unfixed whole blood at 4 degrees C and at room temperature (RT, approximately 20 degrees C), and the leukocytes prepared for analysis using the Coulter Q-Prep system, (ii) leukocytes which had obtained following the removal of erythrocytes from whole blood by dextran sedimentation and which had been washed or left unwashed at RT, and (iii) leukocytes which had been prepared from whole blood that had been formaldehyde fixed immediately following venesection. The amount of fluorescence associated with the cells was determined by flow cytometry. The expression of CD14 was low under all conditions. However the expression of CD11b, CD16 and CDw32 was significantly higher (p less than 0.05) on neutrophils obtained by dextran sedimentation (n = 4) than on cells which had been fixed with formaldehyde ex vivo; the increase in expression was even greater if the cells had been washed. In contrast, the expression of CD13 on formaldehyde fixed cells was higher than on cells which had been labelled at 4 degrees C or at room temperature and was similar to or slightly lower than that on cells obtained by dextran sedimentation. Increasing the time between 10 and 60 min for which the whole blood was incubated with antisera at RT or at 4 degrees C, resulted in progressive increases in the expression of CD11b and CD13. When neutrophils which had been obtained by dextran sedimentation were incubated with unlabelled antibodies to CD16 or CDw32 and FITC labelled antibodies to CD11b there was a marked increase in the expression of CD11b. Altogether these findings indicate that the analysis of functional molecules on neutrophils (which may be rapidly up-regulated during activation) should be performed under clearly defined and controlled conditions. Dual fluorescence studies may, in some circumstances, produce misleading results.
Macey
M G
MG
Department of Haematology, Royal London Hospital, Whitechapel, UK.
Jiang
X P
XP
Veys
P
P
McCarthy
D
D
Newland
A C
AC
D000704:Analysis of Variance; D015703:Antigens, CD / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis*; D018826:CD13 Antigens; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D006801:Humans; D018950:Lipopolysaccharide Receptors; D016177:Macrophage-1 Antigen / Q000032:analysis; D009504:Neutrophils / Q000276:immunology*; D011961:Receptors, Fc / Q000032:analysis; D017452:Receptors, IgG
D015703:Antigens, CD; D000943:Antigens, Differentiation; D015214:Antigens, Differentiation, Myelomonocytic; D000954:Antigens, Surface; D018950:Lipopolysaccharide Receptors; D016177:Macrophage-1 Antigen; D011961:Receptors, Fc; D017452:Receptors, IgG; D018826:CD13 Antigens; D016650:Fluorescein-5-isothiocyanate