Staining kinetics in single cells

Summary Applying hydrodynamic conditions, which certify a negligible influence of convective diffusion, the time-dependent uptake of thionin in lymphocytes, monkey kidney cells, and their separated nuclei was measured spectroscopically. Using fixed cell material the dye transport inside the cell is not hindered due to plasma membrane and cytoplasm. The staining rate depends on the dye concentration, the pretreatment of the cell, and on the electrolyte concentration of the dye solution. The mechanism of dye migration inside the cell is in accordance with a porous matrix model. The diffusion process takes place inside the pores and channels filled with liquid and is modified by adsorption of dye molecules on the walls of the pores. A dynamic reversible equilibrium exists between migrating dye molecules and the binding sites on the pore walls described by the Freundlich adsorption isotherm. The proposed model explains the observed order of reaction of the staining kinetics.     

Adsorption of rutin with a novel β-cyclodextrin polymer adsorbent: Thermodynamic and kinetic study

The adsorption properties toward rutin of a cyclodextrin polymer adsorbent CroCD-TuC 3 have been studied. The adsorption capacity is reduced as temperature and pH of solution rises, but increases with the increase of solvent polarity. Compared with Sephadex? G-15 dextran gel beads, CroCD-TuC 3 shows dramatically higher isosteric enthalpy due to a significant contribution of rutin/β-cyclodextrin inclusion complex formation in CroCD-TuC 3 skeleton. A highlight in our study is that the pore diffusion model has been employed to describe the mass transfer inside the adsorbent pores. It reveals that the diffusion inside the pores is the rate-restricting step in the whole adsorption process. The effective pore diffusivity of rutin in CroCD-TuC 3 calculated is much lower than the diffusivity in diluted solution. The pore diffusion model is an available tool to investigate the profile of mass transfer inside the pores, and provides an effective method to describe adsorption kinetics.     

The Pneumathodes of Laguncularia racemosa: Little Known Rootlets of Surprising Structure, and Notes on a New Fluorescent Dye for Lipophilic Substances

Abstract: Two types of negatively geotropic aerial roots may be observed on the root system of Laguncularia racemosa: pneumatophores with secondary growth, and short-lived pneumathodes which remain in the primary anatomical state. The pneumathodes distinguish themselves by the absence of an epidermis; instead, the outer cortex takes the place of the outermost tissue. This tissue forms a three-dimensional network of rod-like cells and gas spaces. The cell walls contain a lipophilic substance which ensures that the intercellular spaces remain gas-filled during submergence. An uniseriate cellular layer separates the outer and inner cortex. This uniseriate cellular layer, which we term a "pore layer", is characterized by cells with suberized and lignified cell walls and occasional pores among the cells. The pores permit the diffusion of oxygen-rich air from the surface of the pneumathode to the aerenchyma of the inner cortex and the escape of carbon dioxide from the interior of the root. The structure of the differentiated pneumathode originates from frequent cell divisions in the part of the apical meristem where the outer cortex emerges. Because of the pressure thereby exerted on the epidermis and hypodermis, these two cell layers tear and become separated from the outer cortex. Their remnants remain visible at the base of the pneumathode and as an appendage of the calyptra. The function and significance of the pneumathodes for L. racemosa are discussed. An extract of Xanthoria parietina was employed as a new fluorescent dye to stain suberine in cell walls. The staining technique is presented in this paper.     

N-octane diffusivity enhancement via carbon dioxide in silica slit-shaped nanopores – a molecular dynamics simulation

Equilibrium molecular dynamics simulations were conducted to study the competitive adsorption and diffusion of mixtures containing n-octane and carbon dioxide confined in slit-shaped silica pores of width 1.9 nm. Atomic density profiles substantiate strong interactions between CO₂ molecules and the protonated pore walls. Non-monotonic change in n-octane self-diffusion coefficients as a function of CO₂ loading was observed. CO₂ preferential adsorption to the pore surface is likely to attenuate the surface adsorption of n-octane, lower the activation energy for n-octane diffusivity, and consequently enhance n-octane mobility at low CO₂ loading. This observation was confirmed by conducting test simulations for pure n-octane confined in narrower pores. At high CO₂ loading, n-octane diffusivity is hindered by molecular crowding. Thus, n-octane diffusivity displays a maximum. In contrast, within the concentration range considered here, the self-diffusion coefficient predicted for CO₂ exhibits a monotonic increase with loading, which is attributed to a combination of effects including the saturation of the adsorption capacity of the silica surface. Test simulations suggest that the results are strongly dependent on the pore morphology, and in particular on the presence of edges that can preferentially adsorb CO₂ molecules and therefore affect the distribution of these molecules equally on the pore surface, which appears to be required to provide the effective enhancement of n-octane diffusivity.     

Seed-coat dormancy in Grevillea linearifolia: little change in permeability to an apoplastic tracer after treatment with smoke and heat

BACKGROUND AND AIMS: Seeds of Grevillea linearifolia germinate following fire, and have seed-coat dormancy broken by smoke and heat shock. Smoke breaks seed coat dormancy in Emmenanthe penduliflora by altering the permeability of the seed coat to an internal germination inhibitor, which subsequently escapes. This model was tested for in G. linearifolia by investigating the permeability of the seed coat to diffusion of high-molecular-weight compounds, and whether this changed after exposure to fire cues. METHODS: Germination response of the seeds to heat shock, smoke or heat + smoke was tested. Penetration of Lucifer Yellow dye into intact seeds was examined after 24 and 48 h of exposure, and penetration of the dye from the inside of the seed coat outwards was examined after 24 h. Histochemical staining with Nile Red and Acridine Orange was used to locate cuticles, suberin and lignin. KEY RESULTS: Twenty-three per cent of untreated seeds germinated; heat shock and smoke increased germination additively up to approx. 80 % for both cues combined. Lucifer Yellow did not penetrate fully through the seed coat of untreated seeds, whether diffusing inwards or outwards. Three barriers to diffusion were identified. Treatment with heat or smoke slightly increased penetration of the dye, but did not completely remove the barriers. Suberin was identified in secondary walls of exotestal and mesotestal cells, and was absent from primary cell walls. Movement of Lucifer Yellow occurred through the middle lamella and primary cell wall of suberized cells; movement of the dye was impeded where suberin was absent. CONCLUSIONS: Fire cues did not significantly decrease barriers to diffusion of high-molecular-weight compounds in the seed coat of Grevillea, and must be breaking dormancy by another mechanism.     

Restricted diffusion of molecules in porous affinity chromatography adsorbents.

A restricted diffusion model is constructed and solved in order to study the permeability of large adsorbate molecules in the pores of affinity chromatography media, when the adsorbate molecules are adsorbed onto immobilized ligands. The combined effects of steric hindrance at the entrance to the pores and frictional resistance within the pores, as well as the effects of pore size distribution, pore connectivity of the adsorbent, molecular size of adsorbate and ligand, and the fractional saturation of adsorption sites (ligands), are considered. Affinity adsorbents with dilute and high ligand concentrations are examined, and the permeability of the adsorbate in porous networks of connectivity nT is studied by means of effective medium approximation (EMA) numerical solutions. As expected, the permeability of the adsorbate decreases as the size of the adsorbate and/or ligand molecule increases. The permeability also decreases when the fractional saturation of the ligands increases, as well as when the pore connectivity of the network decreases. The dependence of the permeability on the pore connectivity tends to be less marked in adsorbents with concentrated ligand than in porous media with dilute ligand concentration. The conditions are also presented for which the percolation threshold is attained in a number of different systems. The restricted diffusion model and results of this work may be of importance in studies involving the modeling, prediction of the dynamic behavior, design, and control of affinity chromatography (biospecific adsorption) systems employing porous adsorbents. The theoretical results may also have important implications in the selection of a ligand as well as in the selection and construction of an affinity porous matrix, so that the adsorbate of interest can be efficiently separated from a given solution. Furthermore, with appropriate modifications this restricted diffusion model may be used in studies involving the immobilization of ligands or enzymes in porous solids.     

Experimental data to the theory of fat staining

Summary In vitro experiments in respect to the mechanism of fat staining indicated that fat staining is due to adsorption. Thus, staining, as an adsorption process depends on the concentration of the dye solution, on the specific surface of the fat phase and on the temperature.The staining of fats is not directly affected by the degree of unsaturation of fats. The degree of dye uptake is determined at a given temperature and dye concentration by the physical state of fats. Maximum dye uptake occurs at a temperature near to the melting point. It follows, however, of the nature of adsorption that fats also take up dye below their melting point, in a semi-liquid or solid state.The staining of fat mixtures takes place in a similar way. Fat mixtures containing components of high and low melting point take up the greatest amount of dye at a temperature approaching the melting point of the fat mixture. On raising the temperature, the dye uptake decreased. The degree of dye uptake was almost identical with the actual dye uptake of the component of lowest melting point of the system, observed at the given temperature.With 4 Figures in the Text     

Fluctuation of surface charge in membrane pores

Surface charge in track-etched polyethylene terephthalate (PET) membranes with narrow pores has been probed with a fluorescent cationic dye (3,3'-diethyloxacarbocyanine iodide (diO-C2-(3))) using confocal microscopy. Staining of negatively charged PET membranes with diO-C2-(3) is a useful measure of surface charge for the following reasons: 1) the dye inhibits K(+) currents through the pores and reduces their selectivity for cations; 2) it inhibits [3H]-choline+ transport and promotes 36Cl- transport across the membrane in a pH- and ionic-strength-dependent fashion; and 3) staining of pores by diO-C2-(3) is reduced by low pH and by the presence of divalent cations such as Ca2+ and Zn2+. Measurement of the time dependence of cyanine staining of pores shows fluctuations of fluorescence intensity that occur on the same time scale as do fluctuations of ionic current in such pores. These data support our earlier proposal that fluctuations in ionic current across pores in synthetic and biological membranes reflect fluctuations in the surface charge of the pore walls in addition to molecular changes in pore proteins.     

On the stainability of plant cell walls with alcian blue

This work is a continuation of a communication on the stainability of broad bean (Vicia faba L.) root tip cells with alcian blue, published some time ago. Following the standard method of staining with alcian blue, the cell walls are very strongly stained, the nuclei (except nucleolus) lightly, the nucleolus and cytoplasm are practically colourless. The weak dyeing of the nucleus is not equal throughout the whole section so that the comparison of stainability of cell walls and nuclei by itself cannot explain the staining with alcian blue. The results of this work on the staining of cell walls (if not including model experiments and experiments in vitro, which are not considered as decisive here) can be summarized as follows: the pH dependence of staining, the loss of stainability as a result of pectinase digestion, blocking of staining by methylation and regeneration of stainability by demethylation and, finally, the impossibility of staining in the presence of NaCl lead to the conclusion that the staining of the material studied in this work is primarily caused by the salt linkage of alcian blue with the free carboxyls of pectic substances. From the comparison of staining with alcian blue and with other basic dyes it follows that in the case of alcian blue some other factors may also take part and are the reason for the selectivity and firmness (fastness) of the staining of cell walls with this dye. Otherwise, the staining of plant cell walls with alcian blue corresponds quite well to the staining of carboxyls containing polysaccharides of animal tissues with this dye. By staining with alcian blue it was found impossible to distinguish between younger and older cell walls within the meristem. However, this staining is suitable for routine use when studying the meristematic tissue. It is often possible to use solutions of a higher pH than generally used.     

A study of interaction of 7-aminoactinomycin D with DNA by fluorescence correlation spectroscopy

The investigation of interaction of 7-aminoactinomycin D with DNA is hampered at nanomolar concentrations by low quantum yield of the dye fluorescence and high adsorbability of the dye. It was found that, instead of the increase in the fluorescence of 7-aminoactinomycin D after binding with DNA, a decrease in the fluorescence takes place, because 7-aminoactinomycin D complexed with DNA exists in two states: photochemically active and inactive. The presence of the photochemically active state of 7-aminoactinomycin D causes an apparent increase in the diffusion coefficient upon embedding of 7-aminoactinomycin D into DNA. The data obtained allow one to state that 7-aminoactinomycin D: (1) forms dimers at micromolar concentration, (2) has two specific photodestruction times, and (3) binds with DNA more effectively than actinomycin D. Polarization measurements made it possible to estimate numerically the adsorption of 7-aminoactinomycin D on the walls of the measuring cell.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Mass transfer kinetics in the chromatography of insulin variants under nonlinear conditions

Experimental data regarding the thermodynamics and kinetics of adsorption of lispro, an insulin variant, onto a YMC ODS-A column, from an aqueous solution of acetonitrile (31%) and TFA are reinterpreted, using a more complex model of the mass transfer kinetics. The adsorption behavior follows the Toth isotherm model, suggesting either a strongly heterogeneous surface or, rather, that when insulin molecules adsorb they contact the surface along different areas of the molecule. The lumped pore diffusion (POR) model of chromatography accounts well for the band profiles. The internal mass transfer resistances are higher than expected, which suggests that intraparticle diffusion is slower. Furthermore, the pore diffusion coefficient increases with decreasing sample size. That surface diffusion accounts for the mass transfer kinetics inside particles explains these results. Assuming that the gradient of the surface concentration is the driving force of surface diffusion, it is possible to account very well for the band profiles of samples of widely different sizes, using a single value of the surface diffusivity.     

Staining of macromolecules: possible mechanisms and examples

This review is based on a presentation given at the Biological Stain Commission meeting in June 2008. I discuss staining as an interaction between dye, solvent, and biological macromolecules. Most staining takes place in water, where the physico-chemical properties of the macromolecules are particularly important. Staining from aqueous solution is summarized. The first step is diffusion–ion exchange, which builds up the dye ion concentration close to the appropriately charged tissue constituents. While charge interactions are important for selectivity and build-up of dye ions around specific tissue and cell constituents, they have in most cases little to do with actual dye binding. The next step, actual binding, is predominantly between aromatic and other non-polar parts of the dye and corresponding groups in the tissue constituent. This results in a reduction of the total hydrophobic area exposed to water, hence the term hydrophobic interaction. Because dye binding is predominantly by dispersive forces, the larger the aromatic dye system and the fewer the number of charges on the dye, the greater the substantivity or affinity. Some relatively straightforward anionic or cationic one-step staining systems are discussed also. These include amyloid staining with Congo red, elastin staining with orceins, collagen staining with picrofuchsin, DNA–RNA staining with methyl green-pyronin Y, acid heteroglycan staining with Alcian blue, and metachromatic staining.     

Intracellular fluorescent probe concentrations by confocal microscopy.

A general method is described that takes advantage of the optical sectioning properties of a confocal microscope to enable measurement of both absolute and relative concentrations of fluorescent molecules inside cells. For compartments within cells that are substantially larger than the point spread function, the fluorescence intensity is simply proportional to the concentration of the fluorophore. For small compartments, the fluorescence intensity is diluted by contributions from regions outside the compartment. Corrections for this dilution can be estimated via calibrations that are based on the intensity distribution found in a computationally synthesized model for a cell or organelle that has been blurred by convolution with the microscope point spread function. The method is illustrated with four test cases: estimation of intracellular concentration of a fluorescent calcium indicator; estimation of the relative distribution between the neurite and soma of a neuronal cell of the InsP3 receptor on the endoplasmic reticulum; estimation of the distribution of the bradykinin receptor along the surface of a neuronal cell; and relative distribution of a potentiometric dye between the mitochondria and cytosol as a means of assaying mitochondrial membrane potential.     

The reaction between some dyes and synthetic hydroxyapatite 2. Nature of the binding reaction

Synopsis In order to study the reactions involved in some of the histochemical procedures used for demonstrating calcium in calcified tissues, it was considered appropriate to use well characterized synthetic hydroxyapatite in the first instance. In the first paper of this series (Speirs, 1970), it was found that many dyes not previously used in histochemistry were capable of staining hydroxyapatite; the purpose of the present paper is to describe the numerous experimental approaches that have been made in an attempt to elucidate the mechanisms involved in the adsorption of some of these dyes by hydroxyapatite. Dyes have been grouped according to their adsorption curves (in which dye uptake by solid was plotted against the concentration of dye in solution at equilibrium). From these graphs, predictions and calculations were made concerning the orientation of the dye molecules on the surface of hydroxyapatite, the type of bonding possibly involved and the area of surface covered by each molecule. These were then related to the dimensions and structure of the dye molecules. Saturation of surface sites was achieved in the adsorption of some dyes and the nature of these sites was investigated by studying (1) competition between several dyes for the surface, (2) the accessibility of surface calcium and phosphorus in stained and unstained hydroxyapatite, and (3) the release of³²P from surface labelled hydroxyapatite during dye adsorption. Most of the dyes adsorbed from 95% ethanol were displaced relatively easily by treatment with 0.5 mM phosphate in ethanol, but those adsorbed from tris buffer, pH 7.45, were more stable when exposed to phosphate in tris. Treatment of stained hydroxyapatite with solvents containing 0.5 mM calcium reduced the rate of elution of the dyes. Convincing evidence for chelation, hydrogen bonding, ion exchange and physical adsorption processes as the mechanisms of adsorption has not been obtained. Future studies to investigate these processes are discussed.     

The Alcian Blue and combined Alcian Blue-Safranin O staining of glycosaminoglycans studied in a model system and in mast cells

Synopsis Polyacrylamide films containing different glycosaminoglycans have been applied to the study of the Alcian Blue and combined Alcian Blue-Safranin O staining procedures. It was found that the polyacrylamide matrix can be interpreted as some kind of barrier around the substrate molecules, a situation which can be compared to a certain extent with what occursin situ, where complex protein molecules can likewise form a barrier.The Alcian Blue staining of the model films was found to follow the Lambert-Beer law. The time to reach optimal dye binding depended on the concentration of the glycosaminoglycan enclosed in the model films and on the concentration of Alcian Blue in the dye solution. Lowering the pH of the dye solution appeared to increase the rate of staining. Optimal staining of model films in the presence of salt or urea was not possible, because under these conditions the pores of the polyacrylamide matrix became blocked. Alcian Blue was found to bind irreversibly to the glycosaminoglycan molecules enclosed in the polyacrylamide films.The results of the combined Alcian Blue-Safranin O staining applied to model films appeared to be highly dependent on the amount of Alcian Blue bound to the glycosaminoglycan in the first step of the double staining procedure. No specific differences were noticed between the behaviour of the different glycosaminoglycan-Alcian Blue complexes towards the Safranin O binding in the mext step. As the theoretical basis for the application of the combined Alcian Blue-Safranin O staining was also found not to be completely valid, the conclusion was reached that this double staining cannot be used for the histochemical identification of glycosaminoglycans. The colour retained by a certain glycosaminoglycan-containing part of the specimen only delivers information about the accesibility of that part for Alcian Blue.     

Detection of DNA molecules in a lipid nanotube channel in the low ion strength conditions

Investigation of the transport phenomena in the nanoscopic channels/pores with the diameter smaller than 100 nm is of utmost importance for various biological, medical, and technical applications. Presently, the main line of development of nanofluidics is creation of biosensors capable of detecting single molecules and manipulating them. Detection of molecules is based on the measurement of electric current through a channel of appropriate size: when the molecule enters the channel, which diameter is comparable with the molecule size, the ion current reduces. In order to improve transport properties of such channels, their walls are often coated with a lipid bilayer, which behaves as two-dimensional liquid and thus is capable of supporting transport phenomena. In the present work, we utilized this property of lipid membranes for the development of a method for detecting and controlling transport of single-stranded DNA through channels formed by membrane cylinders with the luminal radii of 5–7 nm. We have demonstrated that in the conditions of small ion strength, the appearance of a DNA molecule inside such channel is accompanied by an increase of its ion conductivity and can be controlled by the polarity of the applied voltage. The amplitude of the ion current increase allows evaluating the amount of DNA molecules inside the channels. It was also demonstrated that upon adsorption of DNA molecules on the lipid bilayer surface, the membrane cylinder behaves as a voltage-sensitive selective ion channel.     

Envelope structure of four gliding filamentous cyanobacteria.

The cell walls of four gliding filamentous Oscillatoriaceae species comprising three different genera were studied by freeze substitution, freeze fracturing, and negative staining. In all species, the multilayered gram-negative cell wall is covered with a complex external double layer. The first layer is a tetragonal crystalline S-layer anchored on the outer membrane. The second array is formed by parallel, helically arranged surface fibrils with diameters of 8 to 12 nm. These fibrils have a serrated appearance in cross sections. In all cases, the orientation of the surface fibrils correlates with the sense of revolution of the filaments during gliding, i.e., clockwise in both Phormidium strains and counterclockwise in Oscillatoria princeps and Lyngbya aeruginosa. The lack of longitudinal corrugations or contractions of the surface fibrils and the identical appearances of motile and nonmotile filaments suggest that this structure plays a passive screw thread role in gliding. It is hypothesized that the necessary propulsive force is generated by shear forces between the surface fibrils and the continuing flow of secreted extracellular slime. Furthermore, the so-called junctional pores seem to be the extrusion sites of the slime. In motile cells, these pores exhibit a different staining behavior than that seen in nonmotile ones. In the former, the channels of the pores are filled with electron-dense material, whereas in the latter, the channels appear comparatively empty, highly contrasting the peptidoglycan. Finally, the presence of regular surface structures in other gliding prokaryotes is considered an indication that comparable structures are general features of the cell walls of gliding microbes.     

Removal of Astrazon Yellow 7GL from aqueous solutions by adsorption onto wheat bran

Adsorption kinetic and equilibrium of a basic dye (Astrazon Yellow 7GL) from aqueous solutions at various initial dye concentration (50-300 mg/l), pH (4-10), adsorbent dosage (2-8 g/l), particle size (354-846 microm) and temperature (30-50 degrees C) on wheat bran were studied in a batch mode operation. The result showed that the amount adsorbed of the dye increased with increasing initial dye concentration and contact time, whereas particle size and pH had no significant affect on the amount of dye adsorbed by the adsorbent. A comparison of kinetic models on the overall adsorption rate showed that dye/adsorbent system was best described by the pseudo second-order rate model. The removal rate was also dependent on both external mass transfer and intra-particle diffusion. The low value of the intraparticle diffusivity, 10(-11) cm2/s, indicated the significant influence of intraparticle diffusion on the kinetic control. The adsorption capacity (Q0) calculated from the Langmuir isotherm was 69.06 mg/g for at pH 5.6, 303 K for the particle size of 354 microm. The experimental data yielded excellent fits with Langmuir and Tempkin isotherm equations. Different thermodynamic parameters showed that the reaction was spontaneous and endothermic in nature.     

Pore-to-pore hopping model for the interpretation of the pulsed gradient spin echo attenuation of water diffusion in cell suspension systems

A simplified pore-to-pore hopping model for the two-phase diffusion problem is developed for the analysis of the pulsed gradient spin echo (PGSE) attenuation of water diffusion in the condensed cell suspension systems. In this model, the two phases inside and outside the cells are treated as two different kinds of pores, and the spin-bearing molecules perform hopping diffusion between them. The size and the orientations of those two respective pores are considered, and then the diffraction pattern of the PGSE attenuation may be well simulated. Nevertheless, the intensity of the characteristic peak decreases with increasing membrane permeability, from which the exchange time may be estimated. We then analyze the experimental 1H PGSE results of the erythrocytes suspension system. The water-residence lifetime in the erythrocyte is obtained to be 10 ms, which is the same as that estimated from the two-region approximation. Furthermore, the PGSE attenuation curve of addition of p-Chloromercuribenzenesulfonate (p-CMBS) is also discussed. It predicts that the alignment of erythrocytes will become normal to the magnetic field direction after the addition of p-CMBS, and inspection using a light microscope confirms that result.