Fluorochrome staining of multilamellar liposomes.

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 x magnification). The fluorochrome is seen to be accumulated in the liposomal membranes. Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time. Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.     

Carotenoid-containing unilamellar liposomes loaded with glutathione: a model to study hydrophobic-hydrophilic antioxidant interaction

Unilamellar liposomes are used as a simple two-compartment model to study the interaction of antioxidants. The vesicle membrane can be loaded with lipophilic compounds such as carotenoids or tocopherols, and the aqueous core space with hydrophilic substances like glutathione (GSH) or ascorbate, mimicking the interphase between an aqueous compartment of a cell and its surrounding membrane.

Unilamellar liposomes were used to investigate the interaction of GSH with the carotenoids lutein, β-carotene and lycopene in preventing lipid peroxidation. Lipid peroxidation was initiated with 2,2'-azo-bis-[2,4-dimethylvaleronitrile] (AMVN). Malondialdehyde (MDA) formation was measured as an indicator of oxidation; additionally, the loss of GSH was followed. In liposomes without added antioxidant, MDA levels of 119 ± 6 nmol/mg phospholipid were detected after incubation with AMVN for 2 h at 37°C. Considerably lower levels of 57 ± 8 nmol MDA/mg phospholipid were found when the liposomal vesicles had been loaded with GSH. Upon incorporation of β-carotene, lycopene or lutein, the resistance of unilamellar liposomes towards lipid peroxidation was further modified. An optimal further protection was observed with 0.02 nmol β-carotene/mg phospholipid or 0.06 nmol lycopene/mg phospholipid. At higher levels both these carotenoids exhibited prooxidant effects. Lutein inhibited lipid peroxidation in a dose-dependent manner between 0.02 and 2.6 nmol/mg phospholipid. With increasing levels of lycopene and lutein the consumption of encapsulated GSH decreased moderately, and high levels of β-carotene led to a more pronounced loss of GSH.

The data demonstrate that interactions between GSH and carotenoids may improve resistance of biological membranes towards lipid peroxidation. Different carotenoids exhibit specific properties, and the level for optimal protection varies between the carotenoids.     

A new fluorescence method for alkaline phosphatase histochemistry.

We have developed a new fluorescence method for the histochemical localization of alkaline phosphatase activity. Calcium phosphate deposited at the sites of alkaline phosphatase activity in a Gomori-type reaction are identified by calcium binding fluorochromes. The calcium binding fluorochromes calcein, calcein blue, and xylenol orange were investigated, with each fluorochrome being included in the alkaline phosphatase incubating medium and used in a single-step procedure. Alkaline phosphatase activity was studied in freeze-substituted, resin-embedded human liver and jejunal biopsies, and each fluorochrome produced intense fluorescence of different colors at sites of alkaline phosphatase activity. Calcein, calcein blue, and xylenol orange produced green, blue, and red fluorescence, respectively. Sites of enzyme activity were accurately localized without evidence of diffusion, and there was an absence of non-enzyme-catalyzed binding of any of the fluorochromes to tissue. This fluorescence method, which is particularly suited to investigating the localization and distribution of the activity of different enzymes in the same section, was used to investigate the distribution and co-localization of alkaline phosphatase and aminopeptidase M in human liver and jejunum.     

Metallic Lakes of the oxazines (Gallamin Blue, Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2-3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3-5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

Stain Technology a Bone Stain for Osteoid Seams in Fresh, Unembedded, Mineralized Bone

Fresh, ground, mineralized bone sections 75-100 μ thick are stained 90 minutes or 48 hours in the Bone Stain, a preparation containing fast green FCF, orange G, basic fuchsin, and azure II. Surface stain is then removed by grinding under running water. Sections are washed in 0.1% zephiran chloride (benzalkonium chloride) or in 0.01% mild soap and again washed in tap water, followed with distilled water. Sections are next differentiated in 0.01% acetic acid in 95% methanol, dehydrated in 95% ethanol and 100% ethanol, cleared in alcohol:xylene 1:1, 1:4, 1:9 and 2 changes of xylol, and then mounted permanently in Eukitt's mounting media.

Osteoid seams stain either green to jade green or red to dark red, incompletely mineralized bone red or orange yellow, and the zone of demarcation light green. The walls of lacunae, canaliculae, feathered bone, procedural artifacts and periosteocyte lacunar low-density versions stain red.

The method helps in the differential diagnosis of certain metabolic bone diseases in human biopsy and autopsy material.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes

The formation of age pigment-like fluorescent substances during the lipid peroxidation of model membranes has been studied. Ferrous ion and ascorbate-induced lipid peroxidation of liposomal membranes containing phosphatidylethanolamine led to the formation of fluorescent substances which have characteristics similar to those of compounds derived from the reaction of phosphatidylethanolamine with purified fatty acid hydroperoxides. The fluorescent substances were accumulated in liposomal membranes, whereas thiobarbituric acid-reactive substances formed during lipid preoxidation were immediately released from the liposomal membranes. The thiobarbituric acid-reactive substances free from the membranes were not reactive with amino compounds such as phosphatidylethanolamine in liposomes or glycine in aqueous phase. It was suggested that the products reacting with amino compounds are short-lived, and may be rapidly inactivated after released into aqueous phase. The formation of fluorescent products was inefficient when phosphatidylethanolamine incorporated into the liposomes insensitive to lipid preoxidation was incubated with ferrous ion and ascorbate in the presence of liposomes sensitive to the peroxidation. The results suggest that some products generated from peroxidation-sensitive lipids react with the amino group of phosphatidylethanolamine molecules which are located on the same membranes, forming fluorescent substances. The presence of phosphatidylethanolamine in the membrane suppressed the formation of thiobarbituric acid-reactive substances, suggesting that phosphatidylethanolamine may react with radicals formed and terminate the propagation.     

Fluorescence Microscopy Applied to Entomology and Allied Fields

Fluorescence is a phenomenon observable in many substances including a wide range of biological constituents. By use of ultraviolet illumination and the proper fluorescent dyes, when needed, many details of structure and physiological differentiation are made apparent which by illumination with visible light are obscure.

The fluorescence microscope is a valuable adjunct to the study of fluorescence in biological materials. This instrument is discussed from a practical standpoint. The simplifications in the instrument which do not impair its efficiency are indicated.

The use of fluorochromes is discussed and a list of the most important of these is given. Important technics with the fluorescence microscope, including intravital microscopy, fluorescent photomicrography, and microspectroscopy, are described.     

Surface potential and the interaction of weakly acidic uncouplers of oxidative phosphorylation with liposomes and mitochondria

The pH dependence of the binding of weakly acidic uncouplers of oxidative phosphorylation to rat-liver mitochondria and liposomes is mainly determined by the pK_a of the uncoupler molecule.

The absorption and fluorescence excitation spectra of the anionic form of weakly acidic uncouplers of oxidative phosphorylation are red-shifted upon interaction with liposomal or mitochondrial membranes. The affinity for the liposomes, as deduced from the red shift, is independent of the degree of saturation of the fatty acid chains of different lecithins. The intensity of the spectra at one pH value is strongly dependent upon the surface charge of the liposomes. With positively charged liposomes the results obtained can be almost quantitatively explained with the Gouy-Chapman theory, but with negatively charged ones deviations are observed. At a particular pH, the divalent ion Ca²⁺ strongly influences the intensity of the spectra in the presence of negatively charged liposomes, but has no effect with neutral liposomes.

With mitochondrial membranes an effect of Ca²⁺ similar to that with negatively charged liposomes is observed. Depletion of the phospholipids of the mitochondria and subsequent restoration of the mitochondrial membrane with lecithin, strongly diminishes this effect, but restoration with negatively charged phospholipids does not influence it.

From these observations it is concluded that the anionic form of the uncoupler molecule when bound to mitochondria is located within the partly negatively charged phospholipid moiety of the membrane, with its anionic group pointing to the aqueous solution.     

Mallory's Connective Tissue Stain Following Hematoxylin

The following combination of hematoxylin with Mallory's connective tissue stain is useful in bringing out nuclei as well as in differentiating tissue:

Slightly overstain in Mayer's hematoxylin (50 g. potassium alum and 0.2 g. sodium iodate added to 1 liter 0.1% aqueous hematoxylin). Wash; and stain 30 seconds to 1 minute in 0.04% aqueous acid fuchsin-Stain 4 minutes in: 0.5 g. anilin blue and 2 g. orange G dissolved hi 100 cc. of 1% aqueous phosphomolybdic acid. Pass thru 95% alcohol to absolute; clear in xylol and mount in balsam.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

Surface potential and the interaction of weakly acidic uncouplers of oxidative phosphorylation with liposomes and mitochondria.

The pH dependence of the binding of weakly acidic uncouplers of oxidative phosphorylation to rat-liver mitochondria and liposomes is mainly determined by the pKa of the uncoupler molecule. The absorption and fluorescene excitation spectra of the anionic form of weakly acidic uncouplers of oxidative phosphorylation are red-shifted upon interaction with liposomal or mitochondrial membranes. The affinity for the liposomes, as deduced from the red shift, is independent of the degree of saturation of the fatty acid chains of different lecithins. The intensity of the spectra at one pH value is strongly dependent upon the surface charge of the liposomes. With positively charged liposomes the results obtained can be almost quantitatively explained with the Gouy-Chapman theory, but with negatively charged ones deviations are observed. At a particular pH, the divalent ion Ca-2+ stongly influences the intensity of the spectra in the presence of negatively charged liposomes, but has no effect with neutral liposomes. With mitochondrial membranes an effect of Ca-2+ similar to that with negatively charged liposomes is observed. Depletion of the phospholipids of the mitochondria and subsequent restoration of the mitochrondrial membrane with lecithin, strongly diminishes this effect, but restoration with negatively charged phospholipids does not influence it. From these observations it is concluded that the anionic form of the uncoupler molecule when bound to mitochondria is located within the partly negatively charged phospholiped moiety of the membrane, with its anionic group pointing to the aqueous solution.     

In vitro characterization of a novel polymeric-based pH-sensitive liposome system

This study demonstrates rapid and pH-sensitive release of a highly water-soluble fluorescent aqueous content marker, pyranine, from egg phosphatidylcholine liposomes following incorporation of N-isopropylacrylamide (NIPA) copolymers in liposomal membranes. The pH-sensitivity of this system correlates with the precipitation of the copolymers at acidic pH. In vitro release can be significantly improved by increasing the percentage of anchor in the copolymer and thus favoring its binding to the liposomal bilayer. In the case of liposomes containing a poly(ethylene glycol)-phospholipid conjugate, the insertion of the pH-sensitive copolymer in the liposomal membrane appears to be sterically inhibited. Dye release from these formulations at acidic pH can still be achieved by varying the anchor molar ratio and/or molecular mass of the polymers or by including the latter during the liposome preparation procedure. Removal of unbound polymer results in decreased leakage only when the copolymer is inserted by incubation with preformed liposomes, but can be overcome by preparing liposomes in the presence of polymer. Aqueous content and lipid mixing assays suggest contents release can occur without membrane fusion. The results of this study indicate that the addition of pH-sensitive copolymers of NIPA represents promising strategy for improving liposomal drug delivery.     

Study of the Mode of Action of Some Nitrodiphenyl Ethers

Nitrosoderivatives of the nitrodiphenyl ether herbicides (nitrofen, bifenox) have been studied. UV irradiation in different organic solvents gives degradation products. In buffered aqueous media, in the presence of chloroplasts and spin traps such as DMPO, hydroxy and peroxy radicals have been characterized.

In organic media and in the presence of spin traps such as DMPO, PBN, 4-POBN, solvent radicals (CHCIl₂, CCI₃, CH₂O) have been formed.

Nitro-derivatives have been studied under UV irradiation and in the presence of tetramethylethylene (TME), alkenylhydroxylamines are formed which autoxidize in nitroxide radicals. The formation of the stable nitroxide radical occurs in the dark process after continuous irradiation. The intensity of the signal decreases strongly when a new irradiation is applied. Radical species, with analogous ESR spectral characteristics are formed on reaction with nitrodiphenyl ethers and fatty acids.

The reactivity of these herbicides in micellar media (SDS, Brij 35, and CTAB) has been investigated. The kinetics of formation of the ESR signal corresponding to the photoreduction of the nitrodiphenyl ether in the presence of TME behave differently in a micellar environment as compared to solution. The intensity of the formation of the nitroxide increases under irradiation and decreases in the dark; the rotational correlation time t_c has been determined for each type of micelle.

Synthetic nitrosodiphenyl ether made by the reduction of nitrodiphenyl ether using hydrogen gas and PtO₂ as a catalyst gives the corresponding amine, which is oxidized with rneta-chloroperbenzoic acid (m.CPBA). The nitrosodiphenyl ether in the presence of soja azolectin liposorne containing a fluorescent probe has been analysed. When this synthetic nitrosodiphenyl ether is added to a medium containing soja azolectin liposomes and a carboxyfluorescein, fluorescent probe placed inside the liposornes, a rapid increase in the fluorescence of the medium is observed. The nitrosodiphenyl ether induce a break in the liposorne membrane.     

Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry

BACKGROUND: Phycobiliproteins play an important role in fluorescent labeling, particularly for flow cytometry. The spectral properties of R-phycoerythrin (R-PE) and allophycocyanin (APC) have made them the dominant reagents in this class of fluorochromes. In this study, we evaluate a lesser-known but potentially important series of low-molecular weight cryptomonad-derived phycobiliproteins (commercially termed the CryptoFluortrade mark dyes) for their applicability to flow cytometry, both in extracellular and intracellular labeling applications. METHODS: Several cell lines were labeled with biotin-conjugated antibodies against expressed extracellular surface proteins, followed by streptavidin conjugates of three cryptomonad phycobiliproteins (CryptoFluor-2, CryptoFluor-4, and CryptoFluor-5). Cells were then analyzed by flow cytometry using a variety of laser lines and emission filters to establish the optimal excitation/emission characteristics for each fluorochrome. Some cells were permeabilized and labeled for intracellular antigens, also using the cryptomonad fluorochromes. Where appropriate, parallel samples were labeled with other fluorochromes (including R-PE, APC, the cyanin dyes Cy3 and Cy5, and others) to gauge the performance of the cryptomonad fluorochromes against fluorescent labels previously evaluated for flow cytometry. RESULTS: CryptoFluor-2 possessed excitation/emission maxima similar to those of APC and Cy5, with good excitation in the red (HeNe laser 632 nm) and strong emission in the far red (660 nm). CryptoFluor-4 possessed excitation/emission maxima similar to those of Cy3, with optimal excitation in the green (Kr 530 nm) and strong emission in the yellow/orange (585 nm). CryptoFluor-5 possessed excitation/emission maxima similar to those of lissamine rhodamine, with optimal excitation in the yellow (Kr 568 nm) and emission in the orange (610 nm). All cryptomonad fluorochromes gave satisfactory results for both intracellular and extracellular labeling, with detection sensitivities that were comparable or better than traditional phycobiliproteins and low- molecular weight synthetic fluorochromes such as the cyanin dyes. CONCLUSIONS: The CryptoFluor fluorochromes were applicable to flow cytometric immunodetection, with excitation and emission conditions commonly found on multilaser instruments. Performance of several of these dyes was at least comparable to existing fluorescent labels. The low molecular weights (30-60 kd) of phycobiliproteins may make them particularly useful in intracellular antigen detection. Cytometry 44:16-23, 2001. Published 2001 Wiley-Liss, Inc.     

The permeability of liposomes to nonelectrolytes. I. Activation energies for permeation.

The effect of temperature on the permeability of nonelectrolytes across liposomal membranes above and below their transition temperature has been studied by using an osmotic method. Below their transition temperature, liposomes are osmotically insensitive structures but, on addition of gramicidin A, the water permeability so increased that the permeability of solutes could be studied. The measured activation energies for permeation of a variety of nonelectrolytes has been found to increase when a) there is an increase in the capability of the solutes to form hydrogen bonds in water, b) the cholesterol concentration in the membranes increases and c) the membranes pass from a liquid-crystalline to a solid-crystalline state. The change in the activation energy for permeation per hydrogen bond is about 1.8 kcal/mole for all the different liposome systems investigated; the only solute tested that deviated from this correlation was urea, whose activation energy for permeation across a gramicidin-containing system was much lower than expected from its hydrogen-bonding capacity. This finding suggests that urea is permeating across the gramicidin pore. Although the literature contains only incomplete data relating the activation energies for permeation of nonelectrolytes across biological membranes to their hydrogen-bonding capacity, the available evidence suggests that there is a similar correlation to that found in liposomes. Thus, the average increase in the activation energy per hydrogen bond for permeation across ox red cell membranes (Jacobs, Glassman & Parpart, J. Cell. Comp. Physiol. 7:197, 1935) is 2.2 plus or minus 0.4 kcal/mole, a value that is similar to that obtained in liposomes. However, the activation energies for water and urea are - in such a system - very much lower than expected, suggesting that they, too, are permeating by some parallel route such as an aqueous pore.     

Cholesterol superlattice modulates CA4P release from liposomes and CA4P cytotoxicity on mammary cancer cells

Liposomal drugs are a useful alternative to conventional drugs and hold great promise for targeted delivery in the treatment of many diseases. Most of the liposomal drugs on the market or under clinical trials include cholesterol as a membrane stabilizing agent. Here, we used liposomal CA4P, an antivascular drug, to demonstrate that cholesterol content can actually modulate the release and cytotoxicity of liposomal drugs in a delicate and predictable manner. We found that both the rate of the CA4P release from the interior aqueous compartment of the liposomes to the bulk aqueous phase and the extent of the drug's cytotoxicity undergo a biphasic variation, as large as 50%, with liposomal cholesterol content at the theoretically predicted C(r), e.g., 22.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol for maximal superlattice formation. It appears that at C(r), CA4P can be released from the liposomes more readily than at non-C(r), probably due to the increased domain boundaries between superlattice and nonsuperlattice regions, which consequently results in increased cytotoxicity. The idea that the increased domain boundaries at C(r) would facilitate the escape of molecules from membranes was further supported by the data of dehydroergosterol transfer from liposomes to MβCD. These results together show that the functional importance of sterol superlattice formation in liposomes can be propagated to distal targeted cells and reveal a new, to our knowledge, mechanism for how sterol content and membrane lateral organization can control the release of entrapped or embedded molecules in membranes.     

A Consideration of French's Test of Basic Fuchsin for Endo's Medium

In describing a method of testing for the return of color in decolorized fuchsin for use in Endo Medium, French states that variations in hydrogen ion concentration fail to influence the appearance of color in this medium.

Duplications of this test were made using alcoholic and aqueous solutions of fuchsin and both sodium sulfite and sodium bisulfite as decolorizing agents.

In the decolorized alcoholic solutions of fuchsin the color failed to reappear when formalin was added, but a small amount of a weak solution of lactic acid caused the color to return.

Alcoholic solutions of fuchsin failed to decolorize in sodium bisulfite solutions until a few drops of NaOH were added. The color, then, reappeared immediately.

Solutions of peptones to which fuchsin had been added were substituted for the original fuchsin solution. Alcoholic and aqueous solutions of fuchsin were added to equal amounts of a 1% peptone solution. The peptone solutions varied in their hydrogen ion concentration and the results showed that those which were neutral decolorized readily while the more acid solutions were but partially decolorized.

Fuchsin decolorized according to results found in this test, was not satisfactory in the Endo medium, especially in the case of the aqueous solutions of fuchsin.

Experiments which were carried on by other workers and checked with this method all indicated that some acid is necessary to secure the restoration of color.     

A New Microchemical Reaction for Cellulose

A microchemical test for cellulose applicable to fresh sections and commercial products is described. The test differs from the older technics in that materials tested are not permanently altered.

Two solutions are required: (1) 2% solution of iodine in 5% KI, diluted with 9 parts by volume of water containing 0.28% glycerin; (2) saturated aqueous LiCl.

Procedure: Apply 2 or 3 drops of solution 1 with a glass rod; allow the preparation to stand for 30 sec; blot with filter paper, drying as completely as possible. Apply one drop of solution 2, cover and examine. The color reaction will be obtained within 5 min. The reaction for pure cellulose is light blue. Reactions for 16 fibers are given in the table.

As a stain for demonstrating plant tissues the technic has been used in the Botany Department of Pomona College with much success; but this phase of the subject has not been extensively investigated.     

Detection of very low receptor numbers on cells by flow cytometry using a sensitive staining method

We describe a staining method for flow cytometry that resolves with a high degree of sensitivity very low numbers of cell surface molecules, which are normally too few to detect using the conventional fluorescein-conjugated reagents. We took advantage of the fact that liposomes can be constructed to contain hundreds of thousands of fluorochrome molecules per vesicle; antigen specificity can be conferred by covalently conjugating them to antibodies or protein A. Unlike fluorochromes such as fluorescein isothiocyanate (FITC) that are directly conjugated to protein ligands with a fluorochrome to protein ratio of about 2 to 1 on the average, their large encapsulating capacity gives liposomes a tremendous potential for signal amplification. In an indirect immunofluorescence study using liposomes that contained the fluorochrome carboxyfluorescein (CF) and that were covalently conjugated to protein A, we were able to obtain up to 50 times the fluorescence signal over background that could be detected with FITC-conjugated protein A. Scatchard analysis showed that the thymoma cell line RDM4 expresses 23,000 and 2,600 binding sites for monoclonal antibodies (mAb) against H-2K and H-2D, respectively. When RDM4 cells were treated with anti-H-2K mAb followed by FITC-conjugated protein A, at best we were able to obtain a fluorescence signal that was only 7 times above background. However, when these cells were treated with the same antibody followed by protein A conjugated to small unilamellar liposomes or large unilamellar liposomes, the fluorescence signals were 110 and 335 times above background, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)