Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes

Bouteiller, Philippe P. Le; Mishal, Zohair; Lemonnier, François; Kourilsky, F. M.

doi:10.1016/0022-1759(83)90224-7

Cited by 80 publications

(34 citation statements)

References 25 publications

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“…Since the site of integration was not responsible for the heterogeneity of levels of expression, several other abscissa and ordinate are the same as in Figure 1; b) calculated as described in reference (17); c) calculated as described in Materials and Methods and Figures 3 and 4. possibilities could explain this observation. 1) The amount of @2-m available in these cells and shown as essential for the cell surface expression of HLA heavy chains (1,4,20) might limit the level of expression.…”

Section: Discussionmentioning

confidence: 99%

“…Detailed explanations of the method of calculation relating the number of fluorochrome molecules to the mean number of mAb molecules bound per positive cell, obtained after subtraction of the control values, are given in reference (17). For each sample, 10,000 cells were analysed for fluorescence intensity after exclusion of dead cells by light scatter gating measurements.…”

Section: Calibration Of the Cell Sorter And Quantitativementioning

confidence: 99%

“…Beyond this qualitative analysis there is a need for quantitative data giving the number of molecules expressed, particularly for the study of the regulation of expression. Several methods using flow cytometry that allow such a quantitative analysis have been described (3,9,17,26,34).The aims of this paper are: 1) to define, with a Calibrated cell sorter and monoclonal antibodies (mAb) conjugated to fluorochrome (171, the quantitative levels of expression of HLA class I at the surface of selected cloned transfected L-A3, B7, CW3 cells; 2) to analyse the stability of this expression; and 3) to define whether a correlation existed between the levels of expression and the number of integrated HLA genes. …”

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Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry

et al. 1985

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By using a calibrated cell sorter and monoclonal antibodies conjugated to fluorochromes, a quantitative analysis of the levels of expression of HLA class I molecules at the surface of cloned murine L cells transfected with purified A3, B7, or C W 3 genes was performed and compared with radioimmunoassay data. We selected clones of heterogenous levels of HLA class I expression, which were shown to remain constant over a period of 4 mo in absence of HAT selection and not to be correlated to the DNA copy number of the corresponding integrated gene.Key terms: DNA mediated gene transfer, monoclonal antibodies, HLA class I gene, flow cytometry.Recent technological developments in DNA-mediated gene transfer and flow cytometry (FCM) have made possible novel approaches to the analysis of the expression and regulation of gene products (10,311 such as those of the major histocompatibility complex (6,8,18,32). When total DNA is used for transfection, populations expressing very rare cell surface antigens can be isolated by repeated sortings (lO,ll), which gives valuable material to cloning of corresponding genes.When cloned genes are used, a qualitative analysis of their products expressed at the surface of transfected cells is generally performed (2,15,20,25). Beyond this qualitative analysis there is a need for quantitative data giving the number of molecules expressed, particularly for the study of the regulation of expression. Several methods using flow cytometry that allow such a quantitative analysis have been described (3,9,17,26,34).The aims of this paper are: 1) to define, with a Calibrated cell sorter and monoclonal antibodies (mAb) conjugated to fluorochrome (171, the quantitative levels of expression of HLA class I at the surface of selected cloned transfected L-A3, B7, CW3 cells; 2) to analyse the stability of this expression; and 3) to define whether a correlation existed between the levels of expression and the number of integrated HLA genes. MATERIALS AND METHODS Monoclonal Antibodies (mAb) and Their Conjugation to FluorochromeThe origin and characteristics of the mAb used are listed in Table 1. B9.12.1, B1.23.2, BlG6, 11-4.1, and 10.3.6 mAb were conjugated to fluorescein isothiocyanate (FITC) according to the method described in reference (17). Molar fluorochrome/protein (FP) ratios of FITC-conjugated mAb, determined after correction for quenching (17), were: B9.12.1,2.0; B1.232, 2.0; Bl.lG6, 1.2; 11.4.1,2.3; and 10.3.6,l.O. Transformation of Ltk-cellsCloned Ltk-murine fibroblasts (H-2kf, Iak-) were transfected by the calcium phosphate technique (35), either with cosmids 44 or 64 (HLA-A3) (19), cosmid 42 (HLA-CW3) (19), or cosmid 37 (HLA-B7) (12), all of which contained the herpes virus thymidine kinase (tk) gene. Control HLA-t k + cells were obtained by transfection with pAG0 DNA which contains the tk gene (20). TKt transfectants were selected in HAT medium (35). Transfected cells were cloned once or twice by limiting dilution (average 0.2 cell/well) and maintained in RPMI 1640 medium supplemented with 5% f...

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Section: Discussionmentioning

confidence: 99%

Section: Calibration Of the Cell Sorter And Quantitativementioning

confidence: 99%

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Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry

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“…Diffraction for a Gaussian beam is equal to: [5] 2x IInD @d = -any given moment, the illumination of the particle is uniform. where 1 A1 I is the intensity change due to absorption, I…”

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Fluorescence response and sensitivity determination for ATC 3000 flow cytometer

Gaucher

Grünwald²,

Frelat³

1988

Cytometry

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The fluorescence emitted by labeled particles after interaction with exciting light is conditioned by laser beam geometry and by the mode of fluorescence collection and filtration. A laser elliptic focusing mode is described, and the fluorescence characteristics of the sample cell flow are calculated. Fluorescence collection and detection through optical filters were analyzed, and efficiency was calculated for the ATC 3000 flow cytometer (Odam-Rruker, Wissembourg, France). A mathematical model is proposed for calculation of the fluorescence signal and its fluctuations. The background noise for the ATC 3000 was quantified experimentally using fluorescent microspheres of a known number of bound equivalent fluorescein isothiocyanate (FITC) molecules. These experimental measurements were found to fit the theoretical predictions, thus validating the proposed model.

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Decreased Accessory Cell Function and Costimulatory Activity BY 1, 25‐Dihydroxyvitamin D₃—Treated Monocytes

Rigby

Waugh

1992

Arthritis & Rheumatism

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To characterize the mechanism(s) by which 1,25-dihydroxyvitamin D, (calcitriol) modulates the costimulatory capacity of monocytes, we examined the effect of calcitriol pretreatment of monocytes on their capacity to promote T cell proliferation (accessory cell function). Correlation of calcitriol-dependent changes in monocyte accessory cell function and alterations in phenotype and cytokine production, and the dependence of these changes on cell viability, were studied. Calcitriol pretreatment induced a defect in accessory cell function that was evident with fixed monocytes, suggesting a cell-surface-associated mechanism. Altered accessory cell function did not correlate with changes in HLA-DR antigen expression and was unaffected by concurrent treatment with interferon-y. Calcitriol treatment did not alter either the expression of adhesion molecules or monocytic production of interleukin-lp (IL-1p) or IL-6. Exogenous IL-1 or IL-6 did not overcome the impaired costimulatory activity of calcitriol-treated monocytes. Thus, calcitriol treatment reduces the capacity of monocytes to promote lectin-induced T cell activation at the level of the plasma membrane, perhaps through altered .~

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Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes

Cited by 80 publications

References 25 publications

Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry

Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry

Fluorescence response and sensitivity determination for ATC 3000 flow cytometer

Decreased Accessory Cell Function and Costimulatory Activity BY 1, 25‐Dihydroxyvitamin D₃—Treated Monocytes

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Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes

Cited by 80 publications

References 25 publications

Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry

Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry

Fluorescence response and sensitivity determination for ATC 3000 flow cytometer

Decreased Accessory Cell Function and Costimulatory Activity BY 1, 25‐Dihydroxyvitamin D3—Treated Monocytes

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Decreased Accessory Cell Function and Costimulatory Activity BY 1, 25‐Dihydroxyvitamin D₃—Treated Monocytes