Comparison between flow cytometry and fluorometry for the kinetic measurement of membrane fluidity parameters

Collins, James M.; Grogan, W. McLean

doi:10.1002/cyto.990100108

Cited by 21 publications

(22 citation statements)

References 14 publications

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“…For nonkinetic measurement of anisotropy, the halfwave retarder was aligned vertical (parallel) to the polarization of the laser beam and the anisotropy was previously obtained by us for Hoechst 33258 stained HeLa using a n SLM subnanosecond fluorometer (4800C series, SLM Instruments, Urbana, IL) (Collins and Grogan, 1989) and the value, 0.32, measured in Hoechst stained Friend erythroleukemia cells by Jovin (1979) with a similar static fluorometer. In other experiments designed to determine variability of cells stained for 30 min with 1 FM DPH, anisotropy values of 0.182 z 0.008 (SEM) were obtained, similar to the value of 0.179 reported by Shinitzky and Barenholz, 1978) for fibroblasts.…”

Section: Anisotropymentioning

confidence: 99%

Plasma membrane fluidity gradients of human peripheral blood leukocytes

Collins¹,

Grogan²,

Scott

1990

Journal Cellular Physiology

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The shape of the fluidity gradient of the outer hemileaflet of the plasma membrane of normal, living, human white blood cells was determined using a series of n-(9-anthroyloxy) fatty acid probes where n = 2, 3, 6, 7, 9, 11, 12, and 16, to establish a baseline for future studies on the consequences of various pathological states. Fluorescence uptake and steady-state anisotropy values were obtained with a flow cytometer capable of continuous recording over time of vertical and horizontal emission intensities, with the output of these intensities as calculated anisotropy values. The fluorescence uptake of all of the membrane probes was rapid up to about 15 min. The magnitudes of the uptake of fluorescence was, for the n-(9-anthroyloxy) series, in the order 2 greater than 3 greater than 6 greater than 7 greater than 9 greater than 11 = 12 = 16 for neutrophils, lymphocytes, and monocytes. Anisotropy values were constant from 5 to 30 min after addition of the various probes. The orders of the anisotropy magnitudes, indicative of the shapes of the fluidity gradient, were, for neutrophils, 6 greater than 7 greater than 9 greater than 2 = 3 = 11 = 12 greater than 16, for lymphocytes, 7 greater than 6 greater than 9 greater than 11 greater than 2 = 3 greater than 11 = 12 greater than 16, and for monocytes, 9 greater than 7 greater than 6 greater than 11 greater than 2 = 3 greater than 12 greater than 16. The kinetics of anisotropy from 1 to 5 min after addition of the probes differed for each of the three cell types. Probes with an n-value less than or equal to the maxima (n = 6, neutrophils; n = 7, lymphocytes; n = 9, monocytes) rapidly (1.2 min) reached equilibrium, whereas those probes with n-values greater than the maxima took progressively longer times to equilibrate as n increased. This behavior is consistent with the existence of an energy barrier just below the approximate region sensed by the probes, which would correspond to just below 6AS for neutrophils, 7AS for lymphocytes, and 9AS for monocytes.

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Section: Anisotropymentioning

confidence: 99%

Plasma membrane fluidity gradients of human peripheral blood leukocytes

Collins¹,

Grogan²,

Scott

1990

Journal Cellular Physiology

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“…Significant differences from control cell response (0 ng/mL) were noted with an asterisk (*) at a p < 0.01 level for IL-1 concentrations of 1.0, 10, and 30 ng/mL. cytometer equipped with standard filters as previously described (Collins and Grogan, 1989).…”

Section: Rna Isolation and Northern Blot Analysismentioning

confidence: 99%

PDGF-α Receptor Subunit Expression Down-regulated by IL-1β in Human Periodontal Ligament Cells

et al. 1998

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The responses of cells to the distinct PDGF isoforms have been correlated directly to the relative numbers of specific PDGF receptor subunits on the cell surface. The modulation of PDGF-alpha receptor subunits, the major subunit expressed in human periodontal ligament (PDL) cells, by cytokines present in the periodontal wound site, such as interleukin-1 (IL-1), may be an important factor influencing regenerative outcomes. The purpose of the present study was to examine the effects of IL-1 beta on PDGF-alpha receptor subunit expression in human PDL cells. Primary cultures of human PDL cells were treated with IL-1 beta over a range of concentrations. We assessed PDGF-alpha receptor subunits by examining the mitogenic responses of cells to PDGF-AA, specific binding of 125I-labeled PDGF-AA, immunofluorescent analysis of PDGF-alpha receptor subunits, and PDGF-alpha receptor subunit mRNA levels using Northern blot analysis. The results demonstrate a significant concentration-dependent decrease in 3H-thymidine incorporation in response to PDGF-AA following IL-1 beta treatment (p < 0.001). This decreased response correlated directly with IL-1-induced decreases in 125I-labeled PDGF-AA binding (p < 0.01), the numbers of immunolabeled PDGF-alpha receptor subunits, and in PDGF-alpha receptor subunit mRNA levels. However, when combined with TGF-beta, IL-1 beta did not show additional down-regulation in proliferative response to PDGF-AA or PDGF-alpha receptor subunits beyond that achieved with these factors individually. These experiments identify IL-1 beta, along with TGF-beta, as significant inhibitors of PDGF stimulation in human PDL cells, acting through the down-regulation of PDGF-alpha receptor subunit expression.

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“…Similarly, no significant difference between bulk fluorometry measurements and those from flow cytometry was observed with HeLa cells (8). We abandoned an initial objective of using liposomes as reference material for each experiment, as such reference preparations were not stable and their fluorescence intensities were highly dispersed.…”

Section: Natural Cellular Fluorescencementioning

confidence: 99%

“…Cytometric fluorescence polarisation studies are common with mammalian material (8,11,13,26), but there are only two elementary measures concerning plant cells (5,6).…”

mentioning

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Flow Cytometric Fluorescence Anisotropy of Lipophilic Probes in Epidermal and Mesophyll Protoplasts from Water-Stressed Lupinus albus L

Gantet¹,

Hubac²,

Brown³

1990

Plant Physiol.

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The blue emission anisotropy, r, of two lipophilic probes, diphenylhexatriene (DPH) and its trimethyl-ammonium derivative (TMA-DPH), has been measured in foliar Lupinus albus L. protoplasts for the first time by flow cytometry. Distinctive values have been obtained for protoplasts of epidermal and mesophyll origin, identified by their intensities of chlorophyll fluorescence. Fluorescence microscopy confirmed that TMA-DPH remained in the plasma membrane while DPH penetrated into intracellular lipid domains. Typical emission anisotropy values at 220C for mesophyll and epidermal protoplasts, respectively, were 0.225 and 0.312 with TMA-DPH, and 0.083 and 0.104 with DPH. This indicates that epidermal cells-and notably their plasma membranes (TMA-DPH)-have higher lipid microviscosity and/or more ordered lipid structure. Two lupin genotypes characterized as resistant or susceptible to drought were analyzed with or without 9 days of water stress shown to increase ion leakage from foliar discs. Water stress greatly increased the apparent fluidity, and more so in the susceptible genotype; the effect was more pronounced in the chlorophyll-containing mesophyll cells than in the epidermal cells.The permeability of proteolipidic cell membranes arises from the interaction of their biochemical and biophysical properties (24). The lipid domain of a membrane is more or less fluid and the lipids are ordered with more or less structure (1, 28); such properties appear to affect the hydration control of the membrane (24), lateral heterogeneity and protein conformation (e.g. in microsomal membranes from bean cotyledons) ( 19).Leaf protoplasts have here been studied with two lipophilic fluorescent probes whose emission anisotropy, r, is related in a nonlinear manner to the dynamic property microviscosity (28) and to structural order of the lipid matrix. We use the term 'lipid fluidity' to englobe the two aspects (30). In fact, lipid order dominates the anisotropy coefficient in steadystate measurements, r, through the limiting fluorescence anisotropy, r. (16). Of the two probes used (below), this is less so for TMA-DPH' (25).Protoplasts have been used for polarization studies previously (3,18,31), but the present work is unique in (a) using 'Abbreviations: TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1 ,3,5hexatriene p-toluenesulfonate; DPH, 1 ,6-diphenyl-1,3,5hexatriene; CV, coefficient of variation; S, Siemens.TMA-DPH for plasma membrane localization, (b) obtaining single cell values, notably to characterize Chl-containing and Chl-less cells, and (c) using the gating function of flow cytometry to eliminate debris, broken cells, or blue autofluorescent cells from the final calculations and to minimize any contribution of diffused light. Cytometric fluorescence polarisation studies are common with mammalian material (8,11,13,26), but there are only two elementary measures concerning plant cells (5, 6).Among the pleiotropic effects of drought upon plants, this report concentrates upon the notion of homeoviscosity in the cellu...

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Comparison between flow cytometry and fluorometry for the kinetic measurement of membrane fluidity parameters

Cited by 21 publications

References 14 publications

Plasma membrane fluidity gradients of human peripheral blood leukocytes

Plasma membrane fluidity gradients of human peripheral blood leukocytes

PDGF-α Receptor Subunit Expression Down-regulated by IL-1β in Human Periodontal Ligament Cells

Flow Cytometric Fluorescence Anisotropy of Lipophilic Probes in Epidermal and Mesophyll Protoplasts from Water-Stressed Lupinus albus L

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