Simple method for comparing large numbers of flow cytometry histograms exemplified by analysis of the CD4 (T4) antigen and LDL receptor on human peripheral blood lymphocytes.

Traill, Karine N.; Böck, Günther; Winter, Ute; Hilchenbach, Martin; Jürgens, Günther; Wick, Georg

doi:10.1177/34.9.2426348

Cited by 36 publications

(24 citation statements)

References 2 publications

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“…Cellular fluorescence increased more than tenfold when labelling time was increased from 0 to 2 h, which demonstrates the difference in cellular fluorescence between assays that measure only binding to the receptor (zero labelling time) and assays that measure both binding and uptake, This is consistent with a cycling time of approximately 10 min for LDL receptors (7). For labelling, we used DiI-LDL at a concentration of 10 mg/liter in order to avoid the nonreceptor-mediated uptake of LDL that takes place at higher concentrations (22).…”

Section: Discussionmentioning

confidence: 99%

“…Different assays for the measurement of receptor activity on blood mononuclear cells have been developed ( 1,2, 6,12,18,[20][21][22]. In radioligand assays, radiolabelled LDL is taken up by the cell, and the nonprotein-bound radioactivity subsequently released to the medium is measured (2,12).…”

mentioning

confidence: 99%

“…These assays all allow the diagnosis of FH in homozygotes. Heterozygotes may also be detected (2,6,20,22), but most authors have reported an overlap between healthy subjects and FH patients.…”

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confidence: 99%

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Standardization of a flow cytometric method for measurement of low‐density lipoprotein receptor activity on blood mononuclear cells

et al. 1995

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Flow cytometric methods for measurement of low-density lipoprotein (LDL) receptor activity on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercholesterolemia (FH). However, cellular LDL receptor activities measured in FH heterozygotes may overlap with those of healthy subjects. Analytical variation is probably responsible for some of this overlap. We have examined several technical details that may affect analytical variation. In each analysis, we included one standard and two control cell preparations. These were cells isolated firom three donors and stored in aliquots at -135°C. Use of standard cells reduced between-series analytical variation of the controls by approximately 50%. Preincubationconditions used to induce the maximum number of receptors, the concentration of fluorochrome 1,l'-dioctadecyl-3,3,3' ,3'-tetramethylindocarbocyanineperchlorate (Di1)-LDL, labelling time, and conditions during storage of labelled cells before flow cytometry were also examined in order to reduce analytical variation. Having standardized the assay, we found among 20 healthy subjects a median receptor activity of 100% vs. 51% among 26 patients who fulfilled clinical criteria for FH. However, four of the patients showed distinctly normal receptor activities, which may suggest either the presence of some other biochemical defect or that in vivo dysfunctional receptors may be measured as normal in some patients with our assay. o 1995 Wiley-Liss, I~C .

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Section: Discussionmentioning

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Standardization of a flow cytometric method for measurement of low‐density lipoprotein receptor activity on blood mononuclear cells

et al. 1995

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“…Receptors have been visualized a t the light and electron microscopic levels in studies with insulin labelled with either ferritin (161,Iz5I (51,rhodamine (26,27,29), or fluorescein (2'7). Fluorescence flow cytometry is used increasingly to analyse the interactions of fluorescently labelled ligands and their cell receptors (4,22,28,30,32). Flow cytometry allows the rapid measurement of cellular properties on a cellby-cell basis.…”

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Insulin binding to human cultured lymphocytes measured by flow cytometry using three ligands

Ziegler¹,

Cantin²,

Germain³

et al. 1994

Cytometry

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The binding of insulin to cultured IM-9 human lymphocytes was studied by flow cytometry using FITC-insulin and biotinylated insulins coupled to streptavidin-phycoerythrin (NaB'-biotinylinsulin (B-insulin) and NaB1 -( biotinyl-Eaminocaproy1)insulin (NBC-insulin)). The reference methods were 1251-insulin binding and the insulin-antiinsulin antibody complexes for flow cytometry. There was a close correlation between 1251-insulin binding and increase in fluorescence for B-insulin, NBC-insulin, and insulin-antiinsulin antibody complexes, but not for FITC-insulin. NBC-insulin gave the largest increase in fluorescence (79 f 9 channels) and the the insulin-antiinsulin antibody complexes the smallest (34 2 2 channels) (P < 0.05). FITC-insulin and B-insulin gave similar results: 47 f 6 and 59 f 6 channels. The concentration reducing 1251-insulin binding by 50% was 1.1 x lo-' M for native insulin, 2.7 x lo-' M for B-insulin, 3.3 x lo-' M for NBCinsulin, and 6.6 x lo-' M for FITC-insulin (P < 0.05). Nonspecific binding was low for B-insulin and NBC-insulin but reached 75% for M FITC-insulin. These results suggest that B-insulin and NBC-insulin are suitable ligands for insulin binding studies using flow cytometry. This two-step procedure is easier than the insulin-antiinsulin antibody complex technique. Its poor affinity, specificity, and sensitivity make FITC-insulin less suitable. o

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“…22 The cellular mean fluorescence intensities for each set of data were calculated and cells with fluorescence intensity in the seventy-fifth percentile or higher were scored as strongly positive for L-type channel expression. Although arbitrary, the choice of seventy-fifth percentile for comparative purposes has proved very sensitive when only small differences exist among cells.…”

Section: Evaluation Of Vgcc Responsivenessmentioning

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Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid)

Wang²,

Zhang³

et al. 2013

IJN

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Introduction:In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). Methods and materials: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells' location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells' VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium Green TM -1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. Results: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K + . This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. Conclusion: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

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Simple method for comparing large numbers of flow cytometry histograms exemplified by analysis of the CD4 (T4) antigen and LDL receptor on human peripheral blood lymphocytes.

Cited by 36 publications

References 2 publications

Standardization of a flow cytometric method for measurement of low‐density lipoprotein receptor activity on blood mononuclear cells

Standardization of a flow cytometric method for measurement of low‐density lipoprotein receptor activity on blood mononuclear cells

Insulin binding to human cultured lymphocytes measured by flow cytometry using three ligands

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid)

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