人红细胞膜血型糖蛋白的研究进展

血型糖蛋白(GP)是红细胞膜中主要含唾液酸的跨膜蛋白,有A、B、C和D四种.GPA是MN血型糖蛋白,GPB表达Ss、U血型,GPC、GPD则是Gerbich抗原,四种血型糖蛋白的结构有不同程度的同源性,尤以同类间的同源性程度最高,GPA在防止红细胞之间、红细胞与血管内皮细胞之间的相互作用有重要功能,并在配体诱导下影响红细胞膜的物理性质.GPC是维持红细胞正常形状、正常物理性质的重要因子.GPA和GPC的功能还分别与带3蛋白、带4.1蛋白有关.     

Quantitative flow cytometric analysis of ABO red cell antigens.

A flow cytometry method has been employed to quantitatively compare the expression of A, B and H antigens on various red blood cells (RBC). The H substance was directly labelled by fluorescein-conjugated anti-H lectin and the A and B antigens by indirect staining first with monoclonal anti-A or anti-B antibodies followed by fluorescently, fluorescein (FITC) or phycoerythrin (PE), labelled anti-mouse immunoglobulin (Ig) antibodies. More than a ten-fold difference in cellular fluorescence intensity was found within each sample. Both the percentage and the mean fluorescence of the positive subpopulation for each antigen were determined. Each RBC population was characterized with respect to the expression of A, B or H antigen by a compound mean value that was the calculated product of these two parameters. The results demonstrated a reciprocal relationship between the compound means of A or B and H. The ratio of A/H or B/H was found to be most informative. Homozygotes for A or B had ratios of greater than 200 and greater than 30, respectively, while heterozygotes (AO or BO) had ratios of less than 5. This method could also distinguish between A1 and A2; RBC carrying the A1 phenotype (as determined by agglutination with anti-A1 lectin) showed a higher A/H ratio than those carrying A2. In contrast to the reciprocity in the expression of A (or B) and H found in RBC obtained from different individuals, a direct correlation was found in the expression of these antigens by individual cells within a given population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous assessment of chromatin structure, DNA content, and antigen expression by dual wavelength excitation flow cytometry.

7-aminoactinomycin D (7-AMD) efficiently discriminates between cells in the G0 and G1 phases of the cell cycle (Stokke et al., Cancer Res. 48:6708, 1988). The fluorescence and light scatter of cells stained with 7-AMD, Hoechst 33258 (H33258), and fluorescein isothiocyanate (FITC)-labeled antibodies were measured by dual wavelength excitation flow cytometry (488 nm, ultraviolet). The H33258 fluorescence was found to reflect DNA content in the presence of 7-AMD, although energy transfer caused an approximately 50% reduction in H33258 fluorescence intensity. However, energy transfer was more pronounced in dead cells, permitting exclusion of such cells during analysis. The G0, G1, S, and G2 phases of the cell cycle could be identified in the 7-AMD versus H33258 fluorescence histograms, as was demonstrated with mitogen-stimulated B lymphocytes and a mixture of unstimulated B lymphocytes and a proliferating B-cell line. One hour fixation with paraformaldehyde was compatible with prefixation labeling of surface antigens with indirectly FITC-labeled antibodies as well as postfixation labeling of intracellular antigens. Studies of expression of some surface and nuclear activation-associated antigens confirmed that cell cycle-resolved antigen expression and the time course of appearance of such antigens could be assessed accurately. Phycoerythrin could be used to label a second antigen.     

Fluorescence spectroscopy of H‐ras transfected murine fibroblasts: A comparison with Monte Carlo simulations

Autofluorescence properties of tissues have been widely used to diagnose various types of malignancies. In this study, we measured the autofluorescence properties of H‐ras transfected murine fibroblasts and the counterpart control cells. The pair of cells is genetically identical except for the transfected H‐ras gene. We applied Monte Carlo simulations to evaluate the relative contributions of Rayleigh and Mie scattering effects towards fluorescence in an in vitro model system of normal and H‐ras transfected fibroblasts. The experimental results showed that fluorescence emission intensity was higher for normal cells than the malignant counterpart cells by about 30%. In normal cells, linearity in emission intensity was observed for cell densities of up to 1.0 × 10⁶ cells/ml whereas for transformed cells it was up to 1.4 × 10⁶ cells/ml. Nuclear volume changes give good account for the differences in the intrinsic fluorescence between normal and malignant cells. The Monte Carlo (MC) code, newly developed for this study, explains both predominant experimental features: the large fluorescence intensity differences between the transfected and the corresponding control cells as well as the phenomena of the red shift in the excitation spectra as a function of cell density. The contribution of Rayleigh scattering was found to be predominant compared to Mie scattering. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 132–140, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

ASSOCIATION OF STRAWBERRY YOGURT SENSORY PROPERTIES WITH PRODUCT COMPOSITION BY PROCRUSTES ANALYSIS

The flavor of eight samples of commercial strawberry yogurt was studied by Free-Choice Profile analysis (FCP). Generalized Procrustes Analysis (GPA) applied to FCP allowed differentiation between samples and highlighted flavor attributes responsible for the observed differences. The relation between sensory and physicochemical datasets was studied by means of GPA. Those samples with higher carbohydrate content were perceived as sweeter, having stronger strawberry flavor, and with more dairy and yogurt flavors. Samples with higher titratable acidity, ash and protein content were perceived as more acidic and higher in intensity of "faulty" or "defective" flavors. Higher moisture content was associated with lower intensity of "dairy" flavors (creamy, dairy, and yogurt) and greater intensity of rancid flavor. It is concluded that, though not often used to this end, GPA is a suitable method to study the relationship of sensory and instrumental measurements.     

Correcting for the inner filter effect in measurements of fluorescent proteins in high-cell-density cultures

Fluorescent proteins (FPs), such as green fluorescent protein (GFP) and its variants, are well-developed visible markers for analyzing bioprocesses. Accurate measurement of fluorescence emitted from FPs in whole cells is complicated by the inner filter effect (IFE), which is caused by intracellular light absorption and scattering by cell particles. The IFE causes nonlinearity between fluorescence intensity and fluorophore concentrations in FP-harboring cells and can significantly influence the accuracy of FP-based analysis, especially at high cell densities. A mathematical model based on detection of fluorescence intensity using a fluorescence spectrophotometer was developed to provide a simple correction for the IFE in fluorescence intensity detection in high-density cultures. The parameters of this model were determined in three different FP-harboring bacterial strains to give the “real fluorescence” intensity without the IFE. Using these parameters, accurate analysis of FP-labeled Escherichia coli at high cell density in pure culture and in mixed cultures with fluorescent and nonfluorescent strains was easily and successfully achieved.     

Aberrancy in immunogenicity and cell-surface expression of H-2 antigens on erythrocytes

Immunogenicity for T cell-independent B-cell response assessed by splenic plaque-forming cell (PFC) response and cell-surface expression measured by laser flow cytometry of various class I H-2 antigens on mouse red blood cells (RBC) were compared. It was found that the order of magnitude of both immunogenicity and cell-surface expression on RBC is H-2D^d H-2D^b > H-2K^d, H-2K^b. Furthermore, H-2^d public antigens and H-2L^d antigens were neither immunogenic nor easily demonstrable on RBC. These findings contrasted with poor immunogenicity for PFC response (Nakashima et al. 1982, 1983) and proportionally strong expression of H-2 antigens on lymphoid cells. Immunogenicity and cell-surface expression of H-2D^d antigen on RBC were not shown to be controlled by the action of genes outside H-2D. It was therefore suggested that a number of H-2 antigens, including H-2K^d private, H-2K^b private, and H-2^d public specificities are at least functionally defective on RBC. This is possibly due to the structural characteristics of the antigens. Since immunogenicity and cell-surface expression were in parallel, the expression of H-2 antigens on RBC must be dictated by a subset of B cells whose activity was assessed by PFC response. This finding supports the view that the H-2 molecules display a new category of activity which is different from their ability to activate T cells and depends on their expression on RBC.     

“Natural” chicken antibodies to red blood cells are mainly directed against the B-G antigen,and their occurrence is independent of spontaneous autoimmune thyroiditis

Using an indirect hemagglutination assay we tested sera from 94 healthy normal White Leghorn (NWL) and 117 Obese strain (OS) chickens with spontaneous autoimmune thyroiditis for the presence of natural antibodies against major histocompatibility complex (MHC) antigens expressed on red blood cells (RBC). In both groups older animals had a significantly increased frequency of such antibodies, but the titer was age-independent. OS chickens showed almost the same frequency of natural antibodies as NWL, and a comparison between OS birds with high antithyroglobulin autoantibody (Tg-AAb) titers and those with low titers also did not reveal a significant difference. We conclude that the occurrence of natural antibodies has no relation to Tg-AAb. The specificity of natural antibodies for MHC-encoded antigens was investigated in indirect immunofluorescence tests including absorption experiments with RBC and white blood cells (WBC). This analysis revealed that of 14 MHC-specific sera 13 were reacting with the B-G antigen, which is present on RBC only. One serum reacted with the B-F antigen, expressed on all somatic cells, i. e., both on RBC and WBC.     

Lateral diffusion of antigen receptors artificially incorporated onto B lymphocytes

In the companion paper, we have shown that palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) incorporated onto B lymphocytes can bind DNP antigens and that this binding causes polyclonal B cell activation. In this study we use fluorescence photobleaching recovery (FPR) techniques to examine the lateral diffusion and mobile fractions of antigen-receptor complexes on receptor-decorated B cells as functions of antigen concentration and epitope density. Antigens used in this study are DNP conjugates of polymerized flagellin (DNP-POL) and linear dextran of 2 X 10(6) m.w. (DNP-DEX). The diffusion coefficient observed for antigen bound to artificial receptors decreases monotonically with increased antigen dose and epitope density. When the artificial receptor-bearing cells are labeled with either relatively high concentrations of medium epitope density antigen or high epitope density antigen, a large fraction of antigen-receptor complexes become immobile in the time scale of the experiment. We attribute this behavior to extensive receptor cross-linking by antigen. In parallel with these FPR experiments, we examined the effects of antigen concentration and epitope density on the polyclonal humoral response of receptor-decorated B cells. We found that the response is a function of both antigen concentration and epitope density similar to that seen in natural B cells. The combined results of these experiments show that cell activation results when the diffusion coefficient of the antigen-receptor complex ranges between 10 X 10(-11) cm2 sec-1 and 5 X 10(-11) cm2 sec-1. These values represent threefold and sixfold decreases from the diffusion coefficient of antigen-free receptors, respectively. However, when either a high antigen concentration or epitope density causes a large fraction of antigen-receptor complexes to become immobile, B cells become unresponsive not only to the bound antigen, but also to LPS. Results obtained in this study are very similar to those obtained in a study performed with natural antigen-specific B cells. Therefore, for the responding population of receptor-decorated B cells, it is possible that antigens activate and paralyze these B cells by mechanisms similar to those by which antigens regulate normal B cell responses.     

Cytofluorometric analysis of R-Thy-1. antigens in various rat lymphocytes with different electrophoretic mobility and organ distribution.

Small bone marrow lymphocytes, which had been previously enriched by velocity sedimentation, thymocytes, lymph node cells and spleen cells were electrophoretically separated, stained with fluorescein conjugated rabbit a-rat-Thy-1. globulin and their fluorescence intensities analyzed with a flow cytophotometer. Thy-1. antigens were found in 80% of the bone marrow small lymphocytes showing low electrophoretic mobility (EPM), in all thymocytes, about 80% of which show low and the rest medium to high EPM, and in a few lymph node cells of high EPM. Thy-1. positive cells were not observed in the spleen. All fluorescence intensity histograms obtained were modal and could be properly fitted with normal curves showing coefficients of variation (C.V.) in the range of 20% to 30%. It was observed that the thymocytes of low EPM had an antibody binding affinity significantly different from that of the other stained lymphocytes. Moreover the surface antigen density decreased in the sequence: thymocytes of low EPM, bone marrow lymphocytes of low EPM and thymocytes of high EPM. The fluorescence intensity of stained lymph node cells of high EPM appeared similar to that of thymocytes of high EPM but was not evaluated precisely. Thus the two dimensional cell analysis provided by a combination of EPM and surface fluorescence of Thy-1.+ cells, allows the characterization of different lymphocyte populations which cannot be clearly identified with normal one dimensional techniques. The biological significance of the results is discussed briefly.     

Expression of the asialo GM1 determinant on murine intestinal epithelia.

Murine intestinal epithelial cells were studied by flow cytometric analyses for the expression of lymphocyte-associated membrane antigens. Three lymphocyte antigens were found to be expressed at high density on most nonhematopoietic intestinal epithelial cells. These included major histocompatibility complex class II antigens, the T cell-associated CT carbohydrate determinant, and the asialo GM1 (aGM1) neutral glycolipid. Examination of aGM1 determinant density on epithelial cells, estimated by fluorescence intensity, indicated that aGM1 was expressed at levels equal to those present on lymphoid cells known to be aGM1+. The potential role for lymphocyte antigenic determinants on nonhematopoietic cells of murine epithelia with respect to local regulation of intestinal lymphocytes is discussed.     

Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.     

Development of an optical immunosensor based on the fluorescence of Cyanine-5 for veterinarian diagnostics

A model optical immunosensor was developed to quantify an antibody present in a sample by measuring the fluorescence of Cyanine-5 conjugated with the antibody, using a competitive and a sandwich immunoreaction configuration, with the antigen immobilised in controlled pore glass beads. At pH 2, 94% of the antigen-antibody complex was dissociated, allowing reutilisation. Photobleaching had no effect on the fluorescence. This model system was used to detect Brucella sp. infection and could quantify anti-Brucella sp. antibodies in ovine serum samples in the range from 0.005 to 0.11 mg ml(-1).     

Molecular characterization of fetal antigens on red blood cells of chickens,Japanese quail,and quail-chicken hybrids

The molecular nature of chicken fetal antigen (CFA) and quail fetal antigen (QFA) was studied on embryonic red blood cells (RBCs) of the chicken, the Japanese quail, and the quail-chicken hybrid. Specific immunoprecipitation of radiolabeled membrane proteins followed by electrophoretic separation and autoradiography were used to identify the protein molecules carrying these fetal antigens. CFA was found on molecules of 24, 50, 88, 99, 130, 170, and 220 kd (kilodaltons) in the chicken and hybrid and on molecules of 24, 50, 99, and 170 kd in the Japanese quail. Similarly, quail fetal antigen was associated with 24-, 50-, 99-, and 170-kd molecules in the quail and hybrid and was not detected in the chicken. Partial proteolytic digestion of the 50- and 170-kd molecules isolated from RBCs of all sources showed remarkably similar peptide patterns. Likewise, two-dimensional separation of the CFA-positive and QFA-positive 50-kd molecules from quail RBCs revealed a similar pattern of at least nine isomorphic variants. Sequential depletions of quail embryonic RBC extracts with either anti-CFA or anti-QFA followed by immune precipitation with the reciprocal antiserum suggested that most of the cell surface proteins carrying QFA also have CFA on the same molecules. It is suggested that specific glycosylations of a variety of distinct molecular weight proteins determines the antigenic phenotype characterized as "fetal antigens."     

一种鉴定MNSs红细胞血型单克隆抗体类型的方法的建立

为鉴定MNSs血型单克隆细胞株6D7C9分泌的抗体类型,通过克隆、亚克隆、细胞转染等分子生物学技术建立了血型糖蛋白GPA、GPB的异源表达系统,并将其作为抗原,通过ELISA、Western 印迹法确定6D7C9分泌的McAb.结果显示,RT－PCR技术成功克隆获得了GPA、GPB血型糖蛋白编码基因,通过分别构建其重组逆转录病毒表达载体pEGZ/GPA及pEGZ/GPB,转染包装细胞293T,再感染L929细胞,经zeocin筛选2周后,RT PCR及流式细胞仪分析证实,L929/GPA和L929/GPB转基因细胞中分别有GPA、GPB目的基因的转录和蛋白表达.用稳定高表达GPA、GPB的转基因细胞通过ELISA和Western 印迹法证实单克隆细胞株6D7C9分泌的是抗GPA/GPB McAb.本研究成功地建立了血型糖蛋白GPA、GPB的异源表达系统,为MNSs血型McAb的检测及GPA、GPB蛋白的功能学研究奠定了基础.     

Fluorescence imaging to quantify the fluorescent microspheres in cardiac tissue

To quantify the fluorescent microsphere (FM) content in cardiac tissue, which is an indicative of blood flow, fluorescence imaging of both sides of the pig heart slice was employed. Despite the light scattering inside the tissue and contributions from multiple tissue layers to the total emission, it is shown that the fluorescence intensity at any pixel is proportional to the FM content and the fluorescence image may be transformed to the image of the FM concentration. A convenient standard for the emission‐FM concentration transformation is proposed. The approach has several advantages in comparison with the traditional “digestion & extraction” method such as: non‐destructiveness, high spatial resolution, high throughput, repeatability and simplicity of operation. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant

The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.     

Adsorption of 3H-Fatty Acid Esters of Streptococcal Groups A and E Cell Wall Polysaccharide Antigens by Red Blood Cells and Their Effect on Hemagglutination

The streptococcal group A and E cell wall polysaccharide (PS) antigens were esterified under identical conditions with four fatty acid chlorides (lauroyl, myristoyl, palmitoyl, and stearoyl), varying from 12 to 18 carbon atoms. With group A PS, it was shown that the four resulting esters varied in their ability to sensitize red blood cells (RBC) to agglutination in the presence of specific antiserum. The most active was palmitoyl (16C) followed by myristoyl (14C). The least active was the lauroyl ester (12C). One-tenth as much palmitoyl ester was required as stearoyl group A PS ester. Such variation in the ability to sensitize RBC was not demonstrated with the group E esters, with the exception of the lauroyl ester which was the least active. Removal of N-acetylglucosamine from the esterified and the nonesterified group A PS by enzyme action resulted in a significant loss of serological activity of both antigens. No appreciable difference in the rate or total loss of activity was found in either case. It was demonstrated that both tritium-labeled stearic and palmitic acids and their respective PS esters were adsorbed in significant amounts to RBC. The results indicate that the esterified antigens were adsorbed to the RBC because of the presence of the fatty acid in the PS ester. Attempts to block the receptor sites on the red cell by presensitizing the cells with fatty acid were negative. Likewise, the adsorbed ester did not prevent the uptake of fatty acid at the levels tested. Tritium-labeled esterified group A PS and group E PS were used to show that the amount of antigen required to produce maximal agglutination was the same when cells from the same individual were used, whereas this was not the case when cells from different individuals were used. The amount of antigen required to produce maximal agglutination varied from one batch of sheep RBC to another. Once the optimal concentration of antigen was reached, any additional adsorption did not increase the titer of agglutination.     

Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: Effect of charge,fluidity and antigen-to-lipid ratio

The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens α-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal adjuvant CAF01 composed of cationic dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6′-dibehenate (TDB) or ii) the neutral adjuvant formulation NAF01, where DDA was replaced with zwitterionic distearoylphosphatidylcholine (DSPC). The effect of liposome charge, bilayer rigidity, isoelectric point and antigen-to-lipid ratio was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, intrinsic fluorescence and Langmuir monolayers. The net anionic α-lactalbumin adsorbed onto the cationic liposomes, while there was no measureable attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of α-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized the liposomal surface charge, resulting in reduced colloidal stability and liposome aggregation. Langmuir studies revealed that α-lactalbumin was not squeezed out of DDA monolayers upon compression, which suggests additional hydrophobic interactions.     

Molecular quantification of cell cycle-related gene expression at the protein level

BACKGROUND: Immunofluorescence cytometry of antigen and DNA content provides relative measurements of the cell cycle phase distribution of a specific epitope. Measurement of correlated expression of epitopes on signaling and regulatory proteins will be useful in the study of the complex pathways involved in cell cycle regulation and carcinogenesis. However, to formulate regulatory pathway models, measurements of molecules per cell would be more useful than relative measurements of intensity. Here, we report on a system in which the relationship between molecules and fluorescence is determined for a reference set of cell lines that are then used to directly calculate the number of molecules for unknowns. To demonstrate the process, we calculated the cell cycle phase distribution of SV40 large T antigen (Tag) in the reference cells. METHODS: A set of cell line clones expressing different levels of Tag were isolated. Quantitative Western blots of these cells and purified, recombinant Tag were performed. Cells from the same sample were stained and analyzed by flow cytometry for Tag and DNA. The relationship between molecules and fluorescence was established and calculations were performed for the phase distributions of Tag. RESULTS: The five cell lines had 0.11, 0.27, 1.06, 2.44, and 2.63 x 10(6) molecules of Tag per cell, determined by Western blot. The average coefficient of variation was 10.6%. The relationship of molecules to fluorescence fit a linear equation (r(2) = 0.96) over the range, 0.11 - 2.63 x 10(6) molecules, however, the same equation did not fit the relationship between 0 molecules, defined by isotype staining controls, and the lowest expressing cell line. To calculate the phase distributions of molecules in the lowest cell line, a second linear equation from 0 to 110,000 molecules was used. CONCLUSIONS: This work describes a system where fixed cells expressing various levels of a target antigen quantified by Western blots can be used to standardize flow cytometric measurements of gene expression in absolute terms.