Jan Hed

The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation

Flow cytofluorimetry identifies and quantifies cell markers of different leukocyte subpopulations by combining cytofluorimetry with the differences in the light scattering properties of the leukocytes in mixed populations. In the phagocytic assay, reported in this paper, the experimental conditions were selected in such a way that it was possible to analyse the phagocytic function of granulocytes in peripheral blood without time-consuming cell separation. The percentage of phagocytosing granulocytes was not dependent on the concentration of granulocytes at the selected incubation time and particle (yeast-C3b) concentration. Furthermore, it was possible to adapt a previously described fluorescence quenching technique (FQ method) to differentiate between attachment and ingestion. Crystal violet, originally used in the FQ method, could not be used in this assay due to its lysomotropic effect. Trypan blue at a concentration of 0.25 mg/ml or higher at pH 4.5 showed a plateau effect in fluorescence quenching indicating an effect on attached but not ingested particles. This assay offers a simple technique to screen the functional properties of phagocytic cells in peripheral blood.

Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay

Immune complexes attached to and ingested by human polymorphonuclear (PMN) cells were quantified by cytofluorometry using a fluorescence quenching assay which permits differentiation between attachment and ingestion. The fluorescence intensity decreased after ingestion as a result of the low pH in the phagolysosomes. When extracellular pH was lowered a slight decrease in phagolysosomal pH was detected in macrophages but not in PMN. When measuring total fluorescence, interaction at pH 5.8 for PMN and at pH 4.4 for macrophages is recommended, since the intensity of extra- and intracellular fluorescence are equal under these conditions. Thirty different dyes were tested for dye exclusion and fluorescence quenching of FITC-conjugated yeast particles, and FITC-conjugated IgG. Because of the lysosomotropic effect of basic dyes, acid and direct dyes are preferable as quenching agents. We could not find physical or chemical properties of the dyes that correlated with their quenching effect. Heat aggregated IgG was used as an immune complex analogue in the development of the assay. Trypan blue (0.2 mg/ml) at pH 4.4 was found to be the best quenching agent of extracellular fluorescence when using ingested aggregated IgG. The technique offers a simple method of quantifying ingested protein aggregates and of studying heterogeneity in phagocyte populations.

A new membrane permeabilization method for the detection of intracellular antigens by flow cytometry

This article describes a new cell membrane permeabilization method for the detection of intracellular antigens by immunofluorescence staining and flow cytometry. The number of cells remained unaltered and no cell aggregation or loss of intracellular antigenicity was observed after this permeabilization treatment. A mixed leukocyte population from human peripheral blood was used in this study and the leukocytes were fixed and permeabilized, which permitted monoclonal anti-vimentin antibodies to reach intracellular antigens. The stabilization of cell membranes and preservation of intracellular antigenicity was achieved with paraformaldehyde fixation. This pretreatment prevents cell destruction and subsequent treatment with the detergent n-octyl-beta-D-glucopyranoside results in permeabilization of the cell membrane. The procedure does not alter the expression of cell surface antigens, which is of importance if phenotypic characterization of intracellularly stained cells is to be undertaken. Furthermore, this simple, rapid and reproducible technique makes it possible to detect and distinguish between different human peripheral blood leukocytes without prior purification steps. The leukocyte subpopulations remain well-separated and easily detectable by flow cytometry.

Altered expression of CD11b/CD18 and CD62L on human monocytes after cell preparation procedures

We have investigated the effect of different cell preparation procedures on the surface expression of CD11b/CD18 and CD62L on human monocytes. Both EDTA and heparin anticoagulated blood were used as sources for leukocytes. The monocytes were analysed by flow cytometry in a mixed leukocyte suspensions obtained after ammonium chloride mediated lysis and in mononuclear cell suspension prepared by density gradient centrifugation (Ficoll-Paque) performed both at 4 degrees C and at 20 degrees C. Monocytes from heparinized blood had a higher expression of CD11b/CD18 and a more pronounced inter-individual variation than monocytes from EDTA blood. Monocytes isolated by Ficoll-Paque had a higher degree of ex vivo activation by means of altered expression of CD11b/CD18 and CD62L compared to monocytes prepared by ammonium chloride mediated lysis. This was more pronounced when the isolation procedure was performed at 20 degrees C. Our findings indicate that monocytes prepared by ammonium chloride mediated lysis of EDTA blood and with the cell handling temperature kept at 4 degrees C are exposed to the smallest ex vivo modulation by means of receptor alteration. An awareness of ex vivo modulation by different cell preparation procedures is of importance especially when comparing the expression of functional receptors on leukocytes of disparate origin.

Enumeration of IFN-γ-producing cells by flow cytometry: Comparison with fluorescence microscopy

Abstract A new intracytoplasmic immunofluorescence staining to detect and quantify human interferon-γ (IFN-γ)-producing cells by means of flow cytometry is described. Mononuclear leukocytes, stimulated in vitro to produce IFN-γ, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic IFN-γ was demonstrated by indirect immunofluorescence using IFN-γ-specific mouse monoclonal antibodies. The staining exhibited a very characteristic morphology and was localized in the Golgi apparatus. An excellent agreement between the enumeration of cytoplasmic IFN-γ-positive cells by immunofluorescence microscopy and flow cytometry was noted. However, the latter has the advantage of a standardized control, is less labor consuming and is observer independent.

Quenching of intracellular autofluorescence in alveolar macrophages permits analysis of fluorochrome labelled surface antigens by flow cytofluoremetry

We report a new technique in which the autofluorescence of alveolar macrophages from smokers is quenched by crystal violet. This technique permits immunostaining of surface antigens of these cells and enables the stained cells to be analysed by flow cytofluorometry. The variable solubility of crystal violet makes it important to characterize the crystal violet solution by its quenching properties and not rely on the assumed concentration of dissolved dye. High concentrations of crystal violet lowered the number of cells and gave a decreased amount of surface antigen (CD11b). However, a lower concentration of crystal violet could be used if the cells were fixed with paraformaldehyde (4%) and the membranes were permeabilized with n-octyl-beta-D-glucopyranoside (0.6%). Using a phagocytic model with FITC-conjugated particles we were able to show that this treatment gave an efficient permeabilization of phagolysosomal membranes.

Complement activation according to the alternate pathway by glass and plastic surfaces and its role in neutrophil adhesion

C3-deposition, generated by complement activation according to the alternate pathway, was detected on borosilicate glass slides and polystyrene Petri dishes. The C3-depositions grew peripherally until the entire surface was covered. The deposits were also visualized with scanning electron microscopy and could not be washed away with low-pH (3.5) or high-pH (9.6) buffers. No consumption of complement function was detected indicating a phenomenon restricted to the glass and plastic surfaces. The C3-deposits could mediate an adherence of human neutrophils.

Intracellular and surface distribution of CD9 in human eosinophils

Expression of CD9 is a feature of both eosinophils and platelets. We have investigated the CD9 expression on resting and activated eosinophils with regard to possibly interacting platelets. Mixed leukocytes were obtained from the platelet‐containing (PC) and platelet‐depleted (PD) peripheral blood of healthy donors. A cell membrane permeabilization technique, the FOG method, enabled us to detect the eosinophils as a separate population and permitted flow cytometric analysis of both surface and intracellular antigens. Monoclonal antibodies against CD61 were used to identify platelets. The CD9/CD61 ratio indicated that CD9 on resting eosinophils originates mainly from eosinophils and not from adhered platelets. No difference in CD9 expression was obtained between resting eosinophils in PC and PD blood. However, the expression of CD9 was decreased (p<0.05) on eosinophils in PMA‐activated PD blood but increased (p=0.001) in PC blood, probably due to interacting platelets since CD61 increased simultaneously. In addition, we were able to detect an intracellularly stored pool of CD9 in eosinophils which decreased after activation with PMA. Together these results indicate a translocation of intracellularly stored CD9 to the cell membrane upon activation, probably followed by a subsequent shedding.

Detection of immune deposits in glomeruli: The masking effect on antigenicity of formalin in the presence of proteins

Formalin is known to mask the antigenicity of immune deposits in glomeruli but not of surface immunoglobulins of isolated lymphocytes. We have shown in mice with experimental passive anti-GBM glomerulonephritis that formalin masks the antigenicity of GBM-bound immunoglobulins only if the tissue is fixed before sectioning. The presence of a high concentration of normal bovine serum during fixation of cryostat sections masks the antigenicity of immune deposits, whereas formalin alone has no obvious effect. The same results were obtained with human immunoglobulins (IgG, IgM and IgA) bound to tissue sections. Protease treatment with pepsin and trypsin restored the ability of the immunoglobulins to be stained. The masking effect seems to be due to extensive cross-linking of environmental proteins which prevents fluorescent conjugates reaching their antigens. Methods for detecting immunoglobulins in tissues must, therefore, take into consideration the influence of fixatives not only on epitopes but also on the environment in which the antigenic determinants are localised.

A flow cytometric method to measure the stimulated mobilization and the intracellular pool of the adhesion promoting glucoprotein Mac-1

The receptor for complement factor C3bi (Mac‐1 or CR3) belongs to a complex of leukocyte surface glucoproteins (CD11/CD18) that are essential for chemotaxis and adhesion of human polymorphonuclear leukocytes (PMN). Granulocytes can increase their surface expression of Mac‐1 upon stimulation and it is proposed that this depends on a rapid mobilization of an intracellular pool of Mac‐1. In the present study we describe a cell membrane permeabilization method that enables the detection of the intracellular pool of Mac‐1 in granulocytes by flow cytometry. The method is based on the use of the non‐ionic detergent n‐octyl‐beta‐D‐glucopyranoside (OG) to permeabilize the cell membranes of paraformaldehyde‐prefixed leukocytes. It is shown that fMLP (5 times 10‐7 M)‐treated cells expose 85% of the total detectable amount of Mac‐1 molecules on the surfaces. The method makes it possible to measure the total detectable pool, the efficiency of Mac‐1 mobilization and the in vivo expression of the receptor. This could be of value when evaluating the role of adhesion proteins in the inflammatory response.