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

Halldén, Gunilla; Sköld, C. Magnus; Eklúnd, Anders; Forslid, Jan; Hed, J.

doi:10.1016/0022-1759(91)90108-r

Cited by 41 publications

(29 citation statements)

References 14 publications

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“…To facilitate a flow cytometric analysis, we used a previously described quenching method [18], which makes it possible to reduce the intracellular fluorescence on immunostained cells without loss of antigen expression. Both smokers' and non-smokers' AM were treated in the same manner.…”

Section: Discussionmentioning

confidence: 99%

“…Irrelevant mouse IgG antibodies of the same isotype and concentration were used as controls. The autofluorescence of the AM was then quenched according to a previously published technique from our group, the FOG method, in combination with the quenching dye crystal violet [18]. Briefly, after one wash (3 ml …”

Section: Preparation Of Cellsmentioning

confidence: 99%

“…In addition, metabolic response after in vitro stimulation with the phorbol ester phorbol myristate acetate (PMA) was measured as a functional test. To facilitate flow cytometric analysis of the cells, we used a quenching technique [18], which reduces the intracellular fluorescence without alteration of cell membrane antigens, and therefore permits analysis of fluorochrome-labelled antibodies by flow cytometry. , and present consumption exceeded 15 cigarettes/day for the last 5 years.…”

Section: Introductionmentioning

confidence: 99%

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Chronic smoke exposure alters the phenotype pattern and the metabolic response in human alveolar macrophages

Sköld

Lundahl

Halldén

et al. 1996

Clinical and Experimental Immunology

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SUMMARYSmoking induces a chronic inflammatory process in the lower respiratory tract, where the alveolar macrophages (AM) are the main phagocytes. In the present study, the expressions of different membrane glycoproteins (CD11abc, CD71, CD54, CD14 and CD16) were determined by flow cytometry in AM from smokers and non-smokers after quenching of the intracellular autofluorescence. The metabolic activity of the AM was quantified as a functional test. The expressions of CD11a, CD54 and CD71 were higher in non-smokers' AM than in smokers'. The expressions of CD11b and CD16 were similar between the groups, while the CD11c was higher in smokers' AM compared with nonsmokers'. The expression of CD14 was weak in both groups, therefore there was no clear-cut difference between the background and positively labelled cell populations. The metabolic response after in vitro stimulation with the phorbol ester phorbol myristate acetate (PMA) was higher in non-smokers' than in smokers' AM. Our results indicate that chronic exposure to tobacco smoke influences both the expression of AM membrane antigens and the metabolic activity. AM from non-smokers express a phenotype more related to cell proliferation and an accessory function. In contrast, receptors reflecting adhesion and phagocytosis were unaltered or even increased in smokers' AM. The findings suggest a functional change in the AM population after chronic smoke exposure.

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Section: Discussionmentioning

confidence: 99%

Section: Preparation Of Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chronic smoke exposure alters the phenotype pattern and the metabolic response in human alveolar macrophages

Sköld

Lundahl

Halldén

et al. 1996

Clinical and Experimental Immunology

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“…The cells were then incubated for intracellular staining with fluorescein-conjugated anti-human CD68 antibody and its isotype antibody control for 30 minutes at 238C (R&D Systems). All samples were prepared and analyzed with and without quenching with 0.2% crystal violet (1 min, 48C; Polyscientific, Bayshore, NY) to reduce autofluorescence (39)(40)(41)(42). Cells were then washed, fixed with 2% paraformaldehyde, and acquired by a FACScalibur cytometer (BD Biosciences, Pharmingen, San Jose, CA) using Cell Quest software.…”

Section: Flow Cytometrymentioning

confidence: 99%

Overexpression of Apoptotic Cell Removal Receptor MERTK in Alveolar Macrophages of Cigarette Smokers

Kazeros

Harvey²,

Carolan³

et al. 2008

Am J Respir Cell Mol Biol

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Mononuclear phagocytes play an important role in the removal of apoptotic cells by expressing cell surface receptors that recognize and remove apoptotic cells. Based on the knowledge that cigarette smoking is associated with increased lung cell turnover, we hypothesized that alveolar macrophages (AMs) of normal cigarette smokers may exhibit enhanced expression of apoptotic cell removal receptor genes. AMs obtained by bronchoalveolar lavage of normal nonsmokers (n 5 11) and phenotypic normal smokers (n 5 13; 36 6 6 pack-years) were screened for mRNA expression of all known apoptotic cell removal receptors using Affymetrix HG-U133 Plus 2.0 microarray chips with TaqMan RT-PCR confirmation. Of the 14 known apoptotic receptors expressed, only MER tyrosine kinase (MERTK), a transmembrane tyrosine kinase receptor, was significantly up-regulated in smokers. MERTK expression was then assessed in AMs of smokers versus nonsmokers by TaqMan RT-PCR, immunocytochemistry, Western analysis, and flow analysis. Smoker AMs had up-regulation of MERTK mRNA levels (smoker vs. nonsmoker: 3.6-fold by microarray, P , 0.003; 9.5-fold by TaqMan RT-PCR, P , 0.02). Immunocytochemistry demonstrated a qualitative increase in MERTK protein expression on AMs of smokers. Increased protein expression of MERTK on AMs of smokers was confirmed by Western and flow analyses (P , 0.007 and P , 0.0002, respectively). MERTK, a cell surface receptor that recognizes apoptotic cells, is expressed on human AMs, and its expression is up-regulated in AMs of cigarette smokers. This up-regulation of MERTK may reflect an increased demand for removal of apoptotic cells in smokers, an observation with implications for the development of chronic obstructive pulmonary disease, a disorder associated with dysregulated apoptosis of lung parenchymal cells.Keywords: apoptosis; MER tyrosine kinase; alveolar macrophages MER receptor tyrosine kinase (MERTK), a 110-kD transmembrane protein member of the receptor tyrosine kinase family of cell surface receptors, plays a role in the clearance of apoptotic cells (1-4). The MERTK gene is normally expressed on mononuclear phagocytes, dendritic cells, retinal pigment epithelial (RPE) cells, and reproductive tissue (5-8). In mice, deletion of MERTK results in delayed clearance of apoptotic cells and development of autoimmune disease (1, 9, 10). In humans, mutations in MERTK result in retinitis pigmentosa, a disease characterized by a defect in the RPE phagocytosis pathway, where the RPE cells are unable to ingest the shed apoptotic tips of photoreceptor cells (11)(12)(13)(14).In the lung, the clearance of apoptotic cells is the normal function of alveolar macrophages (AMs), the monocyte-derived pulmonary representative of the mononuclear phagocyte system (15-18). Because phagocytosis of apoptotic cells by mononuclear phagocytes invokes an array of receptors on the phagocyte that interacts with bridging molecules and cell-specific ligands on the apoptotic cells, the control of expression of apoptotic receptors on the phagocyte plays ...

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“…Agents such as trypan blue quench green autofluorescence but emit at 650 nm and above, sacrificing measurements in the red and far red portion of the spectrum (10), and therefore offer no real advantage here. Crystal violet, a quenching agent that has been applied to the study of autofluorescent alveolar macrophages (11,12), also produces high levels of fluorescence at 650 nm and above (Pollice A, unpublished observations). Thus, for older flow cytometry and laser scanning cytometry instruments, where the number of available data channels may be limited, the use of an autofluorescence correction that preserves multiparameter data channels would be highly useful.…”

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confidence: 99%

A simple correction for cell autofluorescence for multiparameter cell‐based analysis of human solid tumors

Smith

Pollice

Emlet

et al. 2006

Cytometry Part B Clinical

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Background: Corrections that have been proposed to minimize the unwanted contribution of cell autofluorescence to the total fluorescence signal often require either specialized instrumentation or the sacrifice of a data channel so as to perform a measurement that can be used to correct for autofluorescence in individual cells. Here we propose a simple cell by cell correction for autofluorescence that is suitable for multiparameter laser scanning cytometry (LSC) studies in human solid tumors that relies on the ratio of mean autofluorescence to mean total cell fluorescence (mean Fl auto /mean Fl total ). This approach assumes a correlation between the autofluorescence component and the total signal in individual cells. This correction does not require specialized instrumentation, and does not sacrifice a data channel in multiparameter studies. A potential disadvantage is that errors may be introduced by the assumption of a correlation between the two components of the total fluorescence signal in individual cells in samples in which no such correlation exists.Methods: Distributions of cell autofluorescence and total Her-2/neu cell fluorescence were obtained separately by LSC in three human breast cancer cell lines and in three samples of primary human lung cancer. In the breast cancer cell lines, autofluorescence measurements and Her-2/neu measurements were also obtained on the same cells.Results: We show that there is a partial correlation between autofluorescence and total Her-2/neu/FITC fluorescence in individual cells in the three breast cancer cell lines. We also show that the results of a ratio-based autofluorescence correction agree with those based on a true cell by cell correction. Computer simulation studies suggest that in samples with no correlation between the autofluorescence component and the true probe/dye fluorescence component, the ratio correction produces robust estimates of the mean true fluorescence signal, with relatively small but systematic underestimates of the coefficient of variation of such measurements under conditions commonly encountered in the measurement of human solid tumors.Conclusions: A simple cell by cell correction for autofluorescence based on the ratio of mean Fl auto to mean Fl total can be applied in cell samples in which there is a correlation between cell autofluorescence and true probe/dye fluorescence in individual cells. In cell samples that lack this correlation, or in which it is not known whether such a correlation exists, this correction can be used with the reservation that there is a systematic but relatively small underestimation of the degree of variability of the measurements. q

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

Cited by 41 publications

References 14 publications

Chronic smoke exposure alters the phenotype pattern and the metabolic response in human alveolar macrophages

Chronic smoke exposure alters the phenotype pattern and the metabolic response in human alveolar macrophages

Overexpression of Apoptotic Cell Removal Receptor MERTK in Alveolar Macrophages of Cigarette Smokers

A simple correction for cell autofluorescence for multiparameter cell‐based analysis of human solid tumors

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