Human fat cell sizing--a quick, simple method.

Two methods were used to determine the mean cell diameters of 37 samples of human adipose tissue, obtained by open or needle biopsy. Method I was the sizing of cells in cell suspensions and Method II was a quick, simple method of sizing cells from fixed sections. The agreement between the two methods was good (r = 0.93, P = less than 0.001). The results using Method II were slightly lower than those using Method I, and a correction factor is suggested. Method II has several advantages over Method I and we propose that it is a suitable method for sizing cells when a quick method with a permanent record is required.     

Electric sizing of platelets stimulated by adenosine diphosphate (ADP)

The platelet-rich plasma stimulated by a low dose of adenosine diphosphate (ADP) was examined by an electric counter, oscilloscopy, an electron microscopy and packed cell volume analysis. The size distribution curve obtained by the electric counter shifted to the left, and the cell volume was decreased. On the oscillograph, those small cells were seen as pulse signals with a regular shape. However, microscopic observation did not find as many microplatelets or fragments. Also, packed cell volume analysis showed no difference between ADP-stimulated and unstimulated samples. On the electronmicrograph, a slight aggregation was seen, with many of the ADP-stimulated platelets exhibiting spiny forms. The shape factors influences strongly the electric sizing, thus, the deformation of platelets may be the reason for the disagreement between the results by electric observation and those by other methods. Our results indicated that the small platelets observed on the size distribution curve do not always reflect the actual small cells.     

Characterization of rat bone marrow lymphoid cells. I. A study of the distribution parameters of sedimentation velocity, volume and electrophoretic mobility

Various cell populations in rat bone marrow were characterized by means of a two dimensional separation using velocity sedimentation and free flow electrophoresis and by electrical sizing of the separated cells. Up to 4.5 mm/hr five different populations with discrete distributions in volume (coefficient of variation 10% to 13%) and sedimentation velocity (coefficient of variation 6% to 10%) were observed. Three of the small sized populations represented lymphocytes and small normoblasts and two of the larger sized populations represented myeloid cells. Almost all of these cells were in the G0/G1 cycle phase. In the faster sedimenting fractions which contained immature myeloid, erythroid and undefined blast cells and two S phase populations, discrete volume distributions were not evaluated. The cell populations with homogeneous volume (particularly the small lymphocytes) showed high density variations which condiserably impair the separation resolution. The cells sedimenting slower than 3.5 mm/hr were further separated by means of free flow electrophoresis into three peaks differing in electrophoretic mobility (EPM). The peaks of low and high EPM contained two populations and the peak of medium EPM contained three populations all characterized by normal volume distributions of uniform coefficient of variation between 11% and 14%. The small cells in the peaks of high and medium EPM were normolblasts and the other cells were lymphocytes. The biological significance of these results is discussed.     

Erythrocyte and ghost cytoplasmic resistivity and voltage-dependent apparent size.

Particle resistivity is explicitly included in the equations relating volume to voltage pulse, in electronic cell sizing or resistive pulse spectroscopy (RPS). It has long been known that in high electric fields cell resistivity decreases as the membrane undergoes dielectric breakdown. At sufficiently high electric field strengths, well past dielectric breakdown, the red cell membrane becomes electrically transparent, or nearly so, and apparent cell size becomes essentially a function of the cytoplasmic resistivity. Electronic cell sizing is traditionally carried out at low electric field strengths, and corrections made for the influence of cell shape by use of the Laplace equation. We find the Laplace solution to be still applicable at very high electric field strengths for purposes of calculating specific cytoplasmic resistivity from RPS measurements. Our value for discocytes, 220 omega X cm, is in good agreement with published results obtained by other researchers using other techniques. We have also applied these same procedures to determine the time course of voltage-dependent resistivity changes in ghosts and intact spherocytes, during the first 5 min after suspension in hypotonic medium. We believe these to be the first explicit calculations of particle specific resistivity from post-dielectric-breakdown apparent size, using traditional electronic sizing techniques.     

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

Comparison of coulter volumes with radiometrically determined intracellular water volumes for cultured cells

Summary During methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells, proliferation is inhibited, morphologic and biochemical changes occur, and giant, often multinucleated, cells form. We have used the increase in cell volume as a marker of the mature syncytiotrophoblastlike phenotype. Uninduced and differentiated BeWo cells are not spherical, and theoretical considerations suggested that deviations in shape could result in significant errors in Coulter volume. To determine if the values obtained by electrical pulse sizing reflected the actual mass of BeWo cells, we have evaluated the relationship between Coulter volumes and intracellular water volumes obtained using a shape-independent estimate for eight cell types. A close correlation (r ²=0.97) was found, indicating that cell volume changes in populations of irregularly shaped cells can be accurately measured using a Coulter instrument. Supported by an operating grant from the National Cancer Institute of Canada. N.S.B. was a recipient of a studentship award from the Alberta Heritage Foundation for Medical Research. C.E.C. is a Senior Research Scientist of the National Cancer Institute of Canada. The McEachern Laboratory is a research facility of the Faculty of Medicine, University of Alberta, and the Cross Cancer Institute, Edmonton, Alberta.     

Electrical determination of viability in saline-treated mouse myeloma cells.

Suspension of mouse myeloma cells in phosphate buffered saline (PBS) induced a significant amount of cell death. The lethal effects of PBS include an increase in cell lysis, a decreased ability of cells to exclude trypan blue, and a decrease in the colony-forming ability of these cells. Dead cells were also detected on a Coulter counter by the increase in the fraction of cells with a smaller electrical size distribution (ESD). Comparing mixtures of live and dead cells by ESD and trypan-blue exclusion showed a high correlation of electrical size with viability (correlation coefficient = 0.98). Sizing of PBS-treated cells by light microscopy suggested that the altered ESD of the PBS-treated cells was due to a downward shift in the volume distribution. Light-scattering experiments also suggested a decrease in the size of cells after PBS treatment. An increase in permeability of the cell membrane may also contribute to these results. We conclude that electrical sizing is an excellent indicator of physical changes that result from PBS-induced cell death, and is an effective method for distinguishing live and dead mouse myeloma cells after PBS treatment.     

A new red cell discriminant incorporating volume dispersion for differentiating iron deficiency anemia from thalassemia minor

A novel red cell discriminant function [MCV2 x RDW/(Hgb x 100)] was compared to six other discriminants in 102 patients with established mild iron deficiency anemia and 33 patients with beta-thalassemia minor. The discriminant incorporates the two key measurements of erythrocyte cell volume distribution, namely the mean (MCV) and standard deviation (RDW), which are known to be helpful for distinguishing between these two frequent causes of microcytic hypochromic anemia. Data used for the learning set to develop the new discriminant were obtained using an electrical impedance automated whole blood analyzer (Coulter S + IV) and were applied as a validation set for six other discriminants. The discriminant was also tested on smaller subsets of the patients groups using data obtained on either an alternate electrical impedance instrument (Sysmex E-5000) or a laser light scattering based system (Technicon H*1). From the comparison it was concluded that use of a discriminant function that incorporates a measurement of red cell volume dispersion results in enhanced accuracy for distinguishing iron deficiency anemia from thalassemia minor.     

The dielectric method of investigating bound water in biological material: An appraisal of the technique

Three independent dielectric methods for the measurement of water of hydration (bound water) in a biological material are described and discussed comparatively. For well-defined aqueous solutions of biological molecules, hydration can be obtained from direct observations made on the δ dispersion or from measurement of the dielectric values of the β dispersion. For whole tissue, however, neither of these two methods is applicable, and to deduce the hydration, it is necessary to use the third technique in which the volume of the hydrated biological particle is obtained by measuring the effect of it on the known dielectric properties of pure water. The hydration can then be calculated by deducting the volume of the anhydrous particle from the experimentally determined volume of the hydrated particle. Owing to possible systemmatic errors the uncertainty in the absolute hydration value associated with this technique is rather larger than that obtained with the other two dielectric methods. For studying the differences between hydration in similar tissues, however, this objection disappears.     

Cell volume changes during apoptosis monitored in real time using digital holographic microscopy

Cellular volume changes play important roles in many processes associated with the normal cell activity, as well as various diseases. Consequently, there is a considerable need to accurately measure volumes of both individual cells and cell populations as a function of time. In this study, we have monitored cell volume changes in real time during apoptosis using digital holographic microscopy. Cell volume changes were deduced from the measured phase change of light transmitted through cells. Our digital holographic experiments showed that after exposure to 1 μM staurosporine for 4 h, the volumes of KB cells were reduced by ~50-60%, which is consistent with previous results obtained using electronic cell sizing and atomic force microscopy. In comparison with other techniques, digital holographic microscopy is advantageous because it employs noninvasive detection, has high time resolution, real time measurement capability, and the ability to simultaneously investigate time-dependent volume changes of both individual cells and cell populations.     

Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (bMSCs) are multipotent and preferred for cell therapy. However, the content of bMSCs is very low. To propagate a large number of primary bMSCs rapidly has become a prerequisite for bMSC study and application. Different methods of isolating and culturing bMSC were used and compared among groups: bMSCs of group A are isolated using direct adherence method and cultured by conventional medium changing; of group B are isolated using direct adherence method and cultured by low volume medium changing; of group C are isolated using density gradient centrifugation and cultured by conventional medium changing; of group D are isolated using density gradient centrifugation and cultured by low volume medium changing. The average population doubling time (PDT), average generation time and the cumulative cell doubling level were calculated for every group. bMSCs cultured with complete medium containing 10, 11 and 15 % FBS were allocated into group a, b and c separatedly. Cell numbers were counted everyday under a microscope, the population doubling level curve was plotted and PDT was calculated. The growth curve of bMSC in group a, b and c was made. Both density gradient centrifugation and direct adherence methods obtained relatively pure bMSCs. A larger quantity of primary bMSCs were obtained by direct adherence. bMSC proliferation was faster when cultured via the low volume medium changing method at a serum concentration of 11 % than the other methods. Isolating bMSC by direct adherence and culturing by low volume medium changing at a serum concentration of 11 % is preferential for bMSC propagation.     

Osmolarity effects on observed insect cell size after baculovirus infection are avoided using growth medium for sample dilution

Rates of cell size increase are an important measure of success during the baculovirus infection process. Batch and fed batch cultures sustain large fluctuations in osmolarity that can affect the measured cell volume if this parameter is not considered during the sizing protocol. Where osmolarity differences between the sizing diluent and the culture broth exist, biased measurements of size are obtained as a result of the cell osmometer response. Spodoptera frugiperda (Sf9) cells are highly sensitive to volume change when subjected to a change in osmolarity. Use of the modified protocol with culture supernatants for sample dilution prior to sizing removed the observed error during measurement.     

Appraisal of the electrical octet method for estimating earthworm populations in arable land

Quantitative methods are needed for the assessment of the size and composition of earthworm communities. A poorly documented electrical sampling method, Thielemann's octet method, was compared with two long‐established methods, formalin extraction and soil hand sorting, in conventional and direct‐drilled wheat cropping systems at two sites with medium to heavy textured soils in Ireland. Under all agronomic conditions tested, the electrical method extracted significantly higher earthworm numbers than formalin, but earthworm biomasses were not significantly different. When used routinely over two years during periods of high earthworm activity, the electrical method yielded community estimates that were comparable in both size and species composition to those obtained by soil hand sorting (25 cm depth), except in recently ploughed land. However, Murchieona minuscule, a minute endogeic species, was underestimated by electrical extraction. It is concluded that the electrical octet method can be a reliable and useful alternative to other dynamic methods for estimating earthworm populations, especially in situations where minimum soil disturbance is desirable.     

低频脉冲电场对胰岛素介导的细胞增殖影响及其作用机理研究

本文以胞外信号分子胰岛素为研究对象,从细胞信号系统与电磁场相互耦合角度,通过MTT比色法和间接免疫荧光技术研究脉冲电场(f=50Hz,τ=20μS,Epp=1V/m)对人肝细胞(L-02细胞株)的增殖能力以及胰岛素与细胞表面受体的专一结合特性的影响。MTT比色分析结果发现在脉冲电场对细胞增殖的直接作用中,处理5、10、20分钟对细胞增殖的抑制百分率分别为10.13％、18.10％和11.85％;脉冲电场对细胞增殖的间接作用中,胰岛素经脉冲电场处理20、40分钟,对细胞增殖的抑制百分率分别为10.31％和14.12％;流式细胞术检测结果表明细胞悬液和胰岛素共同经脉冲电场处理20分钟组其平均荧光强度降低22.74％,仅胰岛素受电场处理组和仅细胞受电场处理组的平均荧光强度分别下降12.96％和10.51％。本实验结果显示 脉冲电场对人肝细胞的作用体现在两个方面:一是电场直接作用于细胞膜上的胰岛素受体及其他膜成分;二是脉冲电场还可通过改变培养基中的胰岛素分子的构像,影响胰岛素与受体的结合能力,进而影响细胞的增殖等活动。     

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

The computer analysis of volume distribution curves. Demonstration of two erythrocyte populations of different size in the young guinea pig and analysis of the mechanism of immune lysis of cells by antibody and complement.

Guinea pig, rat and sheep erythrocytes were sized electrically using the hydrodynamic focusing technique. The experimental curves were approximated with a computer by linear and logarithmic normal distributions. Rat and guinea pig erythrocytes from adult animals were best approximated by one linear normal distribution. Two populations (I, II) of erythrocytes with different mean volume could be demonstrated in young guinea pigs by this analysis. Population I erythrocytes are small, have a lower electrophoretic mobility and are mainly present at birth. They are gradually replaced by the larger population II erythrocytes. Both types of erythrocytes are probably the result of separate differentiation pathways. The analysis of erythrocyte volume distribution curves during immune lysis by antibody and complement shows that intact and ghost erythrocytes are measured by electrical sizing. No volume changes were observed up to the EAC1-8 intermediate. After the addition of C9, a C9 dose-dependent part of the erythrocytes swell permanently to spheroids. The spheroid transformation is a temperature-dependent, all or nothing reaction which is independent of protein osmotic forces from the interior of the cell.     

Hypertonicity-induced cation channels rescue cells from staurosporine-elicited apoptosis

Cell shrinkage is one of the earliest events during apoptosis. Cell shrinkage also occurs upon hypertonic stress, and previous work has shown that hypertonicity-induced cation channels (HICCs) underlie a highly efficient mechanism of recovery from cell shrinkage, called the regulatory volume increase (RVI), in many cell types. Here, the effects of HICC activation on staurosporine-induced apoptotic volume decrease (AVD) and apoptosis were studied in HeLa cells by means of electronic cell sizing and whole-cell patch-clamp recording. It was found that hypertonic stress reduces staurosporine-induced AVD and cell death (associated with caspase-3/7 activation and DNA fragmentation), and that this effect was actually due to activation of the HICC. On the other hand, staurosporine was found to significantly reduce osmotic HICC activation. It is concluded that AVD and RVI reflect two fundamentally distinct functional modes in terms of the activity and role of the HICC, in a shrunken cell. Our results also demonstrate, for the first time, the ability of the HICC to rescue cells from the process of programmed cell death.     

Evaluation of the electrical potential on the membrane of the extremely alkaliphilic bacterium <Emphasis Type="Italic">Thioalkalivibrio</Emphasis>

The electrical potential on the membrane was measured in cells of strains AL2 and ALJ15 of the extremely alkaliphilic bacterium Thioalkalivibrio versutus using the penetrating cation tetraphenylphosphonium (TPP⁺) and a TPP⁺-selective electrode. The potentials were -228 ± 5 and -224 ± 5 mV, respectively, i.e. higher than in most alkaliphilic bacteria. Membrane potential in the cells was estimated by measuring the inner cell volume by two independent methods: (1) estimation of total cell volume by light microscopy and (2) estimation of the inner aqueous volume of the cells with allowance for the distribution difference of tritium labeled water penetrating through the membranes and a nonpenetrating colored protein. The inner cell volume was 2.4 ± 0.2 and 2.2 ± 0.1 μl/mg of cell protein by the two methods, respectively. Computer computation was used as an alternative to manual calculation to count the number of cells for estimation of total cell volume.     

Detection of isolated tumor cells in peripheral blood and in BM: evaluation of a new enrichment method

Cell enrichment methods that deal with larger volumes of peripheral blood and BM are needed for increased sensitivity of detection, characterization and quantification of isolated tumor cells (ITC). This study was designed to evaluate a new procedure, the RosetteSep-Applied Imaging Rare Event (RARE) detection method, which depletes the majority of the erythrocytes and leucocytes in a peripheral blood (PB) sample, thereby negatively enriching tumor cells if present. This enrichment procedure allows for increased sensitivity, by analyzing a 5-10 fold larger volume of blood, compared with a direct immunocytochemical (ICC) technique, with minimal impact on laboratory workload. Model experiments showed comparable tumor cell recoveries between the two tested methods, both in PB and BM. Clinical samples were evaluated using paired PB and BM samples from 95 carcinoma patients. Analysis of PB results showed that 25.3% had > or = 1 tumor cell detected by the RARE procedure, compared with 5.2% after direct ICC analysis, analyzing a 10-fold larger volume by the RARE procedure. The direct ICC analysis of BM from the same patients revealed 16.8% positive. The ITC detection differed both quantitatively and qualitatively between BM and PB, as samples with high numbers of ITC in BM were still negative in PB. The clinical significance of ITC in blood still needs to be established. However, the easy access of peripheral blood, and the increased sensitivity obtained by increasing the sample volume with the RARE procedure, suggests that the value of peripheral blood analysis should be tested in parallel in studies where ITC detection in BM is performed.     

Measurement of cardiac output by electrical impedance plethysmography

There are many potential applications for cardiac output measurement in clinical and experimental medicine. The most commonly used techniques are invasive procedures, requiring cardiac or arterial catheterization, a disadvantage that has restricted their wider application. Impedance plethysmography has been developed as a non-invasive, beat-by-beat method of cardiac output measurement, which provides an estimate of stroke volume from changes in the electrical impedance of the thorax during cardiac systole. The values for cardiac output obtained by this technique have been extensively compared with values obtained by other methods, both in experimental animals and in the human subject. In the majority of studies high correlation coefficients have been obtained, although impedance plethysmography has tended to give higher absolute values than most other methods. Values for cardiac output obtained by impedance plethysmography are best assessed by comparison with a series of normal values obtained by this technique, rather than with values obtained by other methods. We discuss the results of an investigation of normal cardiac output by impedance plethysmography; theoretical objections to impedance cardiography are considered, and various methods of determining the specific resistivity of blood are reviewed.