Use of a FACS III for fluorescence depolarization with DPH

We have previously demonstrated age-related differences in human lymphocyte membrane fluidity, by use of steady-state polarization measurements on bulk cell suspensions with the fluorescence probe DPH. However, for exact analysis of the possible functional importance of these changes, single-cell measurements were deemed of interest. We have now used an analog division device to measure fluorescence depolarization "p" of DPH in real time with a FACS III flow cytometer. The measurements are reliable, as we have been able to confirm the differences in DPH "p" between monocytes and lymphocytes previously shown in bulk suspension and to demonstrate the expected differences in fluidity of lipid-modulated cells. We also found significant differences in DPH "p" between lymphocytes of young and elderly blood donors. Lymphocyte subsets did not differ in polarization values but did differ in fluorescence intensity with Th less than Ts less than B = NK cells.     

DPH标记细胞膜的动力学与膜脂流动性的荧光偏振校正测量

用稳态荧光技术测得经过校正的荧光成分,由此算出用DPH标记的细胞膜的偏振度。方法是作荧光偏振值在随时间变化的曲线,将其外推至零标记时间求出该时间的荧光偏振值。用此法测定了艾氏腹水癌细胞的膜流动性。结果表明流动性比用整个细胞测得之值小,说明膜脂的有序程度和包装密度比胞浆中的脂大。实验结果和用三房空模型分析所得的理论值符合较好,提示荧光探剂的标记过程主要受分子扩散所控制。     

Heat-induced changes in the membrane fluidity of Chinese hamster ovary cells measured by flow cytometry.

Flow cytometry was used to measure the fluorescence polarization of the lipid probe trimethylammonium-diphenylhexatriene as an indicator of plasma membrane fluidity of Chinese hamster ovary (CHO) cells heated under various conditions. Fluorescence polarization was measured at room temperature about 25 min after heating. When cells were heated for 45 min at temperatures above 42 degrees C, fluorescence polarization decreased progressively, signifying an increase in plasma membrane fluidity. The fluorescence polarization of cells heated at 42 degrees C for up to 55 h was nearly the same as for unheated control populations, despite a reduction in survival. The fluorescence polarization of cells heated at 45 degrees C decreased progressively with heating time, which indicated a progressive increase in membrane fluidity. The fluorescence polarization distributions broadened and skewed toward lower polarization values for long heating times at 45 degrees C. Thermotolerant cells resisted changes in plasma membrane fluidity when challenged with subsequent 45 degrees C exposures. Heated cells were sorted on the basis of their position in the fluorescence polarization distribution and plated to determine survival. The survival of cells which were subjected to various heat treatments and then sorted from high or low tails of the fluorescence polarization histograms was not significantly different. These results show that hyperthermia causes persistent changes in the membrane fluidity of CHO cells but that membrane fluidity is not directly correlated with cell survival.     

Modifications of erythrocyte membrane fluidity of in vivo functionally aged human erythrocytes

The process of red blood cell senescence in the blood stream results in many changes in their physical and biochemical properties. In this work we have studied the physico-chemical state of erythrocyte membranes prepared from 5 subpopulations of erythrocytes of different age by using the fluorescence technique. Membrane fluidity has been evaluated by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and a further study of the fluorescence decay of this probe has been performed by multifrequency phase and modulation fluorometry. DPH fluorescence polarization is significantly increased in the membranes prepared from the youngest fraction of erythrocytes, indicating a decreased fluidity without any significant change in DPH fluorescence decay.     

Use of fluorescence anisotropy determinations for indicating the physiological status of hybridoma cell cultures

The evolution of lipid compartment fluidity during culture of hybridoma cells was studied by fluorescence polarization measurements. The probe partition between the plasma membrane and intracytoplasmic compartments was determined by a quenching fluorescence method. A progressive decrease of the plasma membrane fluidity was observed during the growth phase with an increase during stationary and degeneration phases of the culture. These data suggest that fluidity parameters could be used to follow the behaviour of hybridoma cell cultures.     

Trypanosoma brucei: a fluorescence and spin label study of the membranes of the bloodstream form

Fluidity of the plasma membrane of Trypanosoma brucei brucei has been examined with fluorescence and electron spin resonance spectroscopy. Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 8-anilino-1-naphthalene sulfonate and the spin label probe 5-doxyl stearate have been employed to examine fluidity under a variety of conditions. The temperature dependence of 8-anilino-1-naphthalene sulfonate polarization and of the order parameter S for 5-doxyl stearate reveals phase alterations near 30 C. 1,6-Diphenyl-1,3,5-hexatriene polarization shows that proteolysis of the surface glycoprotein with trypsin increases fluidity but treatment with human serum which is trypanocidal produces no detectable change in membrane fluidity.     

Comparative study on fluorescent probes distributed in human erythrocytes and platelets

Important cellular functions, such as rheological properties of cells are presumably related to the membrane lipid fluidity which may be approached by the use of fluorescence polarization method. However, biological membranes represent very heterogeneous media and the knowledge of the fluidity of membrane compartments requires the use of different probes. Two fluorescent probes, DPH and its cationic derivative, TMA-DPH, have been employed to probe the lipid fluidity of human platelets and red cell membranes. The results show that the informations given by DPH and TMA-DPH can present important differences, suggesting that DPH and TMA-DPH are localized in different regions of cell membranes. In an attempt to investigate relations between lipid fluidity and rheological properties of red cells, the behavior of probes was studied in a "Couette" viscometer with a device for studying the emissive properties of probes when red cell membranes are under shear conditions.     

Resolution of plasma membrane lipid fluidity in intact cells labelled with diphenylhexatriene

The partitioning of fluorescence probes into intracellular organelles poses a major problem when fluorescence methods are applied to evaluate the fluidity properties of cell plasma membranes with intact cells. This work describes a method for resolution of fluidity parameters of the plasma membrane in intact cells labelled with the fluorescence polarization probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The method is based on selective quenching, by nonradiative energy transfer, of the fluorescence emitted from the plasma membrane after tagging the cell with a suitable membrane impermeable electron acceptor. Such selective quenching is obtained by chemical binding of 2,4,6-trinitrobenzene sulfonate (TNBS), or by incorporation of $\text{N-}\text{bixinoyl}$ glucosamine (BGA) to DPH-labelled cells. The procedures for determination of lipid fluidity in plasma membranes of intact cells by this method are simple and straightforward.     

Effect of Exogenous Cholesterol on the Fluidity of Mitochondrlal Membran from Rice Root Apex

The effect of exogenous cholesterol on the fluidity of mitochondrial membrane from riceroot apex was investigated using the DPH fluorescece probe (1,6-diphenyl-l, 3, 5,-hexatriene).Results showed that exogenous cholesterol, either being absorbed by root system during water culture of rice or being added directly to the prepared mitochondrial of control rice root apex, decreased the fluorescence intesity of probe in the mitochondrial membrane and reduced the fluorescence polarization as well as micro-viscosity, but increased the fluidity of the mitochondrial membrane in rice root apex.     

Pitfalls in measuring fluorescence polarization in mitogen-stimulated T-lymphocytes

Fluorescence polarization measurements with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to detect changes in the fluidity of plasma membranes from T-lymphocytes stimulated with mitogens. When the cells were incubated with succinyl-concanavalin A an increase in fluorescence polarization was observed. This, however, could be shown to be due to the interaction of the mitogen with the label DPH and did not reflect changes in the plasma membrane. In purified plasma membranes a decrease rather than an increase of fluorescence polarization was observed.     

外源胆固醇对水稻根端线粒体膜流动性的影响

应用荧光探剂 1,6-二苯基-1,3,5-己三烯(DPH)观察外源胆固醇对水稻极端线粒体膜流动性的影响。结果表明,无论外源胆固醇通过水培根系吸收或是直接添加给予水稻根端线粒体,都能使DPH与线粒体结合后的荧光强度减弱,使荧光偏振度和微粘度降低,增加水稻根端线粒体膜流动性。     

Phagocytosis and membrane fluidity: application to the evaluation of opsonizing properties of fibronectin

The uptake of particles by phagocytic cells involves an increase in the membrane fluidity determined by steady-state fluorescence polarization. Binding and endocytosis of target particles is in vivo enhanced by humoral factors called opsonins. In this work, fluorescence polarization was used to detect in vitro the opsonic activity of a plasma protein: fibronectin. The assay is based on the analysis of membrane fluidity variations following the uptake of gelatinized latex beads by phagocytic cells in the presence or in the absence of fibronectin. Using TMA-DPH as fluorescent probe, it was observed that the increase in membrane fluidity was enhanced in presence of fibronectin and depended upon the enhanced in presence of fibronectin and depended upon the opsonic activity was related to the integrity of the molecule. Using this method, the opsonic activity of various plasmas could be also determined.     

Effects of local anesthetics on membrane properties. I. Changes in the fluidity of phospholipid bilayers.

The effect of the local anesthetic dibucaine on the solid to liquid-crystalline phase transition in phospholipid vesicles was studied by calorimetry and fluorescence polarization. The partition coefficient (greater than 3000) of dibucaine in the membranes of vesicles prepared from acidic phospholipids was more than 20 times higher than in neutral phospholipid membranes under the same conditions. Calorimetric measurements on vesicles prepared form acidic phospholipids (bovine brain phosphatidylserine; dipalmitoylphosphatidylglycerol) showed that dibucaine (1 with 10(-4) M) produced a significant reduction in the gel-liquid crystalline transition temperature (Tc). This fluidizing effect of dibucaine on acidic phospholipid membranes was even more marked in the presence of Ca2+. In contrast, dibucaine at the same concentration did not alter the Tc of neutral phospholipids (dipalmitoylphosphatidylcholine). Significant increase in the fluidity of neutral phospholipid membranes occurred only at higher dibucaine concentrations (2 with 10(-3) M). Measurements of the fluorescence polarization and lifetime of the probe, 1,6-diphenylhexatriene, in acidic phospholipid vesicles revealed that dibucaine (1 with 10(-4) M) caused an increase in the probe rotation rate indicating an increase in the fluidity of the phospholipid membranes. A good correlation was obtained between fluorescence polarization data on dibucaine-induced changes in membrane fluidity and calorimetric measurements on vesicles of the same type.     

The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membranes.

The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions.     

Incorporation of sterol into chloroplast thylakoid membranes and its effect on fluidity and function

The sterol, cholesteryl hemisuccinate, has been incorporated into isolated thylakoid membranes of pea and lettuce chloroplasts in order to modify the fluidity of the lipid matrix. Changes in fluidity have been monitored using fluorescence polarization of the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene and the electron-spin-resonance, spin-label probe, 5-doxyl stearate. Both methods indicate that incorporation of increasing levels of sterol reduces the fluidity of the thylakoid lipid matrix. At room temperature the thylakoid lipid matrix is relatively fluid and the effect of increasing the viscosity is to inhibit partially the maximum rate of steady-state electron flow and reduce the dark rate of reduction of flash-oxidised cytochrome f. The results are discussed in terms of lipid fluidity influencing the rate of lateral diffusion of reduced plastoquinone from photosystem II to photosystem I.     

Age and sex related changes of plasma membrane fluidity in isolated rat hepatocytes

The influence of sex and age on membrane fluidity, has been investigated in 6, 12, 18 weeks old Sprague-Dawley rats. Fluorescence polarization (P) was determined at 37 degrees C with a Perkin Elmer MPF 44A fluorescence spectrophotometer. The fluorescent probe TMA-DPH was added to isolated hepatocytes prepared by collagenase method. The membrane fluidity was constantly lower in males than in females, but the difference was statistically significant only in the 12 weeks old group. Major differences appeared related to aging with a significant age-related decrease in fluidity in all animals.     

胶原、壳聚糖包覆的小单层脂质体的体外稳定性评价

为了提高小单层脂质体（ＳｍａｌＵｎｉｌａｍｅｌａｒＶｅｓｉｃｌｅｓ,ＳＵＶ）的体外稳定性,以５（６）－羧基荧光素（５（６）－ＣＦ）为荧光探针,研究了脂质体被胶原蛋白和壳聚糖这两种天然生物大分子包覆后的通透性。发现,胶原蛋白、壳聚糖和磷脂酰胆碱（ＰＣ）的重量比分别为２：１和８：１时,已能明显降低脂质体的通透性。并以一种分子内电荷转移化合物３－甲氧基－４＇－Ｎ,Ｎ－二甲氨基黄酮（ＤＭＭＦ）为探针,用荧光偏振法研究了胶原蛋白、壳聚糖和ＰＣ在上述比例下,对脂质体膜流动性的影响,发现脂质体在被包覆前后,其膜的流动性没有明显的改变。可见,胶原蛋白、壳聚糖包覆脂质体能够在基本不干扰脂质体膜流动性的情况下,明显地提高其体外的稳定性     

Effect of nicotinic acid on microviscosity in mixed liposomal system of lecithin and sphingomyelin

In rat liver plasma membrane, the molar ratio of sphingomyelin and phospholipid is approximately 1:4, whereas, the molar ratio of phospholipid and cholesterol is 3:1. Considering this ratio to be typical for a real biological membrane, we have studied the effect of anticholesterol and the vasodialatory drug nicotinic acid (NA) on the fluidity profile of a liposomal system of lipids mixed in this ratio using the fluorescence polarization probe 1,6-diphenyl-1-1,3,5-hexatriene. The study reveals that when NA is added to the aqueous dispersion of the mixed lipid system (molar ratio of lipid:NA, 1:1) it creates a more fluid environment for the probe molecule and modifies the fluidity profile of the cholesterol-incorporated liposomal system by eliminating the effect of cholesterol to some extent. The drug also affects the activation energy of diffusion of this system. These results on fluidity have been compared with those in cases of liposomes of individual lipids. The effect of NA on fluidity may be attributed to a mechanical interaction of the drug molecules with the lipid molecules.     

Modulation of (Na+,K+)-ATPase activity by the lipid bilayer examined with dansylated phosphatidylserine

The fluorescent probe 8-(dimethylamino)naphthalene-1-sulfonylphosphatidylserine (Dns-PS) was incorporated into purified lamb kidney Na+- and K+-stimulated adenosinetriphosphatase (EC 3.6.1.3) [(Na+,K+)-ATPase] by using a purified phospholipid exchange protein. Phospholipase C was used to reduce phospholipid content. Up to 40% of the phospholipid could be hydrolyzed with only 10% inhibition of the (Na+,K+)-ATPase, but when 67% of the phospholipid was hydrolyzed, the enzyme was inhibited 53%. To examine the effect of protein on the phospholipid bilayer, the fluorescent parameters of the probe incorporated into the enzyme preparation were contrasted with the same parameters for the probe incorporated into the total lipid extract of the preparation. The polarization of fluorescence of the probe in the lipid extract was 0.118 while in the enzyme preparation it was 0.218. This reflected a decrease in fluidity of the glycerol region of the phospholipid bilayer which was mediated by the protein. This effect increased as the phospholipid content of the (Na+,K+)-ATPase preparation was reduced so that with maximal phospholipid reduction the polarization of fluorescence was 0.262. The protein caused a decrease in the transition temperature from gel to fluid states of the bilayer detected by polarization of the probe. The midpoint temperature transition of the enzyme preparation decreased from 33 degrees C when all phospholipids were present to 20 degrees C when 67% of the phospholipids were hydrolyzed. This decrease was not observed for the lipid extract of these samples. A direct correlation between the (Na+,K+)-ATPase specific activity and the polarization of fluorescence of Dns-PS was found. The reduction in phospholipid content did not affect the steady-state level of phosphorylation of the enzyme by ATP but did affect the rate of dephosphorylation which would require conformational changes of the enzymes. The data showed that the fluidity of the phospholipid bilayer can modulate the activity of the (Na+,K+)-ATPase.     

Membrane fluidity of Toxoplasma gondii: a fluorescence polarization study

Toxoplasma gondii membrane fluidity was investigated by fluorescence polarization. We used 1,6-diphenyl 1,3,5-hexatriene (DPH) as a fluorescent hydrophobic probe. Fluorescence anisotropy (r) and degree of order (s) showed high fluidity properties. Chemical analysis was performed on this parasite. We found a low cholesterol/phospholipid ratio, many unsaturated fatty acids chains, and high phosphatidylcholine and low sphingomyelin amounts. These results were in good agreement with the observed high fluidity. This may be related to the great adaptability of Toxoplasma gondii in infesting a wide variety of host cells.