Measurement of CD2 expression levels of IFN-alpha-treated fibrosarcomas using cell tracking velocimetry

METHODS: A methodology and a mathematical relationship have been developed that allow quantitation of the expression levels of cellular surface antigens, in terms of antibody binding capacities (ABC). This methodology uses immunomagnetically labeled cells and calibration microbeads combined with cell tracking velocimetry (CTV) technology to measure magnetophoretic mobilities corresponding to cellular ABC. The mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads and cells in a nearly constant magnetic energy gradient. RESULTS: Transformed fibrosarcoma cells were given controlled treatments of interferon-alpha in order to manipulate CD2 antigen expression levels. These cells were then immunomagnetically labeled with anti-CD2 FITC antibodies and anti-FITC MACS paramagnetic nanoparticles. Measured magnetophoretic mobilities were used to calculate ABC for these cells, corresponding to CD2 expression levels. CONCLUSION: The results from CTV and flow cytometry (FCM) qualitatively verify that these fibrosarcoma cells express elevated levels of CD2 molecules with increasing interferon-alpha treatment from 0 to 24 h. The mean basal CD2 expression level, in terms of ABC, was calculated to be 27,000 from CTV analysis, whereas FCM indicates a comparable ABC value of 33,000.     

Magnetophoretic mobilities correlate to antibody binding capacities

METHODS: A methodology and a mathematical theory have been developed, which allow quantitation of the expression levels of cellular surface antigens using immunomagnetic labels and cell tracking velocimetry (CTV) technology. RESULTS: Quantum Simply Cellular (QSC) microbeads were immunomagnetically labeled with anti-CD2 fluorescein isothiocyanate (FITC) antibodies and anti-FITC MACS paramagnetic nanoparticles. Magnetophoretic mobility has been defined as the magnetically induced velocity of the labeled cell or microbead divided by the magnetophoretic driving force, proportional to the magnetic energy density gradient. DISCUSSION: Using computer imaging and processing technology, the mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads in a nearly constant magnetic energy gradient. A calibration curve correlating the measured magnetophoretic mobility of the immunomagnetically labeled microbeads to their antibody binding capacities (ABC) has been obtained. CONCLUSION: The results, in agreement with theory, indicate a linear relationship between magnetophoretic mobility and ABC for microbeads with less than 30,000 ABC. The mathematical relationships and QSC standardization curve obtained allow determination of the number of surface antigens on similarly immunomagnetically labeled cells.     

Characterization of antibody binding to three cancer-related antigens using flow cytometry and cell tracking velocimetry

Proper antibody labeling is a fundamental step in the positive selection/isolation of rare cancer cells using immunomagnetic cell separation technology. Using either a two-step or single-step labeling protocol, we examined a combination of six different antibodies specific for three different antigens (epithelial specific antigen, epithelial membrane antigen, and HER-2/Neu) on two different breast cancer cell lines (HCC1954 and MCF-7). When a two-step labeling protocol was used (i.e., anti-surface marker-fluoroscein-isothiocyanate [FITC] [primary Ab], anti-FITC magnetic colloid [secondary Ab]) saturation of the primary antibody was determined using fluorescence intensity measurements from flow cytometry (FCM). The saturation of the secondary antibody (or saturation of a single-step labeling) was determined using magnetophoretic mobility measurements from cell tracking velocimetry (CTV). When the maximum magnetophoretic mobility was the primary objective, our results demonstrate that the quantities necessary for antibody saturation with respect to fluorescence intensity were generally higher than those recommended by the manufacturer. The results demonstrate that magnetophoretic mobility varies depending on the types of cell lines, primary antibodies, and concentration of secondary magnetic colloid-conjugated antibody. It is concluded that saturation studies are a vital preparatory step in any separation method involving antibody labeling, especially those that require the specificity of rare cell detection.     

Binding affinities/avidities of antibody-antigen interactions: quantification and scale-up implications

Bioaffinity interactions have been, and continue to be, successfully adapted from nature for use in separation and detection applications. It has been previously reported that the magnetophoretic mobility of labeled cells show a saturation type phenomenon as a function of the concentration of the free antibody-magnetic nanoparticle conjugate which is consistent with other reports of antibody-fluorophore binding. Starting with the standard antibody-antigen relationship, a model was developed which takes into consideration multi-valence interactions, and various attributes of flow cytometry (FCM) and cell tracking velocimetry (CTV) measurements to determine both the apparent dissociation constant and the antibody-binding capacity (ABC) of a cell. This model was then evaluated on peripheral blood lymphocytes (PBLs) labeled with anti CD3 antibodies conjugated to FITC, PE, or DM (magnetic nanoparticles). Reasonable agreements between the model and the experiments were obtained. In addition, estimates of the limitation of the number of magnetic nanoparticles that can bind to a cell as a result of steric hinderance was consistent with measured values of magnetophoretic mobility. Finally, a scale-up model was proposed and tested which predicts the amount of antibody conjugates needed to achieve a given level of saturation as the total number of cells reaches 10(10), the number of cells needed for certain clinical applications, such as T-cell depletions for mismatched bone marrow transplants.     

Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system.

BACKGROUND: The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. METHODS: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. RESULTS: From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. CONCLUSIONS: As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process.     

Separation of a breast cancer cell line from human blood using a quadrupole magnetic flow sorter.

We have developed a quadrupole magnetic flow sorter (QMS) to facilitate high-throughput binary cell separation. Optimized QMS operation requires the adjustment of three flow parameters based on the immunomagnetic characteristics of the target cell sample. To overcome the inefficiency of semiempirical operation/optimization of QMS flow parameters, a theoretical model of the QMS sorting process was developed. Application of this model requires measurement of the magnetophoretic mobility distribution of the cell sample by the cell tracking velocimetry (CTV) technique developed in our laboratory. In this work, the theoretical model was experimentally tested using breast carcinoma cells (HCC1954) overexpressing the HER-2/neu gene, and peripheral blood leukocytes (PBLs). The magnetophoretic mobility distribution of immunomagnetically labeled HCC1954 cells was measured using the CTV technique, and then theoretical predictions of sorting recoveries were calculated. Mean magnetophoretic mobilities of (1-3) x 10(-4) mm(3)/(T A s) were obtained depending on the labeling conditions. Labeled HCC1954 cells were mixed with unlabeled PBLs to form a "spiked" sample to be separated by the QMS. Fractional recoveries of cells for different flow parameters were examined and compared with theoretical predictions. Experimental results showed that the theoretical model accurately predicted fractional recoveries of HCC1954 cells. High-throughput (3.29 x 10(5) cells/s) separations with high recovery (0.89) of HCC1954 cells were achieved.     

Cell tracking velocimetry as a tool for defining saturation binding of magnetically conjugated antibodies.

BACKGROUND: Continuous flow immunomagnetic separation is an attractive alternative to current batch mode immunomagnetic separation methods because it is capable of high sorting speeds at mild cell conditions, and grants the operator better control of separation process. The control of the separation is dependent on knowledge of the amount of magnetic label attached to the cell (magnetic labeling intensity), however. Determination of the magnetic labeling is accomplished by measuring cell magnetophoretic mobility using a newly developed technique of Cell Tracking Velocimetry (CTV). METHODS: Flow cytometry was used to define the antibody binding characteristics of a fluorescently tagged primary antibody. Subsequently, CTV was used to measure antibody-binding characteristics of a magnetically tagged secondary antibody. RESULTS: The results of this study show that CTV is capable of providing valuable information concerning the cell labeling by magnetically tagged antibodies. It was demonstrated that the magnetically conjugated antibody binding curve exhibits the same exponential increase to saturation characteristics as that seen with the fluorescently tagged antibody. Further, it was shown that the intensity of the secondary magnetic labeling is directly proportional to the intensity of the primary fluorescent label. CONCLUSIONS: CTV is an accurate tool for evaluation of magnetically conjugated antibodies. The ability to determine the intensity of magnetic labeling is necessary for the development of continuous flow immunomagnetic separations based on cell magnetophoresis.     

Evaluation of eluents from separations of CD34+ cells from human cord blood using a commercial, immunomagnetic cell separation system

Human CD34+ cells from cord blood were separated in a two-step process using a commercial, immunomagnetic cell retention system. The performance of the system was evaluated by analyzing a number of eluents from the separations with a number of analytical techniques. In addition to cell counts and flow cytometry analysis, a new experimental technique that is undergoing development, cell tracking velocimetry (CTV), was used. CTV measures the degree to which a cell is immunomagnetically labeled, known as the magnetophoretic mobility, of a population of cells on a cell-by-cell basis and presents the results in the form of a histogram similar to flow cytometry data. The average recovery and purity of CD34+ cells from 10 separations was 52% and 60%, respectively. CTV analysis indicated that the mean magnetophoretic mobility of the positively enriched CD34 cells was 9.64 x 10(-5) mm3/T-A-s, while the mean mobility from negative eluents was -2.02 x 10(-6) mm3/T-A-s, very similar to the mobility of unlabeled cells. Within the positive eluents, the range of magnetophoretic mobility was approximately 50-fold, representing a plausible 50-fold range in surface CD34 antigen expression. CTV analysis also indicated that in some separations, positive cells were not retained by the immunomagnetic cell retention system. Finally, preliminary studies indicate that monocytes might be a primary cause in the lower purities and recoveries seen in this study. It is suggested that the monocytes phagocytose the magnetic nanobeads and become sufficiently magnetized to be retained within the Miltenyi column, reducing the purity of the positive eluent.     

Magnetophoretic cell sorting is a function of antibody binding capacity

Antibody binding capacity (ABC) is a term representing a cell's ability to bind antibodies, correlating with the number of specific cellular antigens expressed on that cell. ABC allows magnetically conjugated antibodies to bind to the targeted cells, imparting a magnetophoretic mobility on each targeted cell. This enables sorting based on differences in the cell magnetophoretic mobility and, potentially, a magnetic separation based on the differences in the cell ABC values. A cell's ABC value is a particularly important factor in continuous magnetic cell separation. This work investigates the relationship between ABC and magnetic cell separation efficiency by injection of a suspension of immunomagnetically labeled quantum simply cellular calibration microbeads of known ABC values into fluid flowing through a quadrupole magnetic sorter. The elution profiles of the outlet streams were evaluated using UV detectors. Optimal separation flow rate was shown to correlate with the ABC of these microbeads. Comparing experimental and theoretical results, the theory correctly predicted maximum separation flow rates but overestimated the separation fractional recoveries.     

Blood progenitor cell separation from clinical leukapheresis product by magnetic nanoparticle binding and magnetophoresis

Positive selection of CD34+ blood progenitor cells from circulation has been reported to improve patient recovery in applications of autologous transplantation. Current magnetic separation methods rely on cell capture and release on solid supports rather than sorting from flowing suspensions, which limits the range of therapeutic applications and the process scale up. We tested CD34+ cell immunomagnetic labeling and isolation from fresh leukocyte fraction of peripheral blood (leukapheresis) using the continuous quadrupole magnetic flow sorter (QMS), consisting of a flow channel (SHOT, Greenville, IN) and a quadrupole magnet with a maximum field intensity (B(o)) of 1.42 T and a mean force field strength (S(m)) of 1.45 x 10(8) TA/m(2). Both the sample magnetophoretic mobility (m) and the inlet and outlet flow patterns highly affect the QMS performance. Seven commercial progenitor cell labeling reagent combinations were quantitatively evaluated by measuring magnetophoretic mobility of a high CD34 expression cell line, KG-1a, using the cell tracking velocimeter (CTV). The CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) showed the strongest labeling of KG-1a cells and was selected for progenitor cell enrichment from 11 fresh and 11 cryopreserved clinical leukapheresis samples derived from different donors. The CD34+ cells were isolated with a purity of 60-96%, a recovery of 18-60%, an enrichment rate of 12-169, and a throughput of (1.7-9.3) x 10(4) cells/s. The results also showed a highly regular dependence of the QMS performance on the flow conditions that agreed with the theoretical predictions based on the CD34+ cell magnetophoretic mobility.     

Continuous flow magnetic cell fractionation based on antigen expression level

Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry.     

Isolation and characterization of the CD133+ precursors from the ventricular zone of human fetal brain by magnetic affinity cell sorting

A fast and effective method to enrich large number of neural precursors from the ventricular zone of human fetus by magnetic affinity cell sorting (MACS) is reported. After incubation with phycoerythrin (PE)-conjugated anti-CD133 antibodies and anti-PE magnetic beads followed by one cycle of MACS, CD133(+) cells were harvested at 85% purity as confirmed by flow-cytometry and immunocytochemistry. In contrast to CD133(-) cells, these CD133(+) cells initiated primary and secondary neurospheres in culture, and the progeny of sorted cells could be differentiated into both neurons and glia, indicating that these highly enriched cells are capable of self-renewal and multi-lineage potential.     

Application of magnetic particle tracking velocimetry to quadrupole magnetic sorting of porcine pancreatic islets

Magnetic isolation is a promising method for separating and concentrating pancreatic islets of Langerhans for transplantation in Type 1 diabetes patients. We are developing a continuous magnetic islet sorter to overcome the restrictions of current purification methods that result in limited yield and viability. In Quadrupole Magnetic Sorting (QMS) islets are magnetized by infusing superparamagnetic microbeads into islets' vasculature via arteries that serve the pancreas. The performance of the islet sorter depends on the resulting speed of the islets in an applied magnetic field, a property known as magnetophoretic mobility. Essential to the design and successful operation of the QMS is a method to measure the magnetophoretic mobilities of magnetically infused islets. We have adapted a Magnetic Particle Tracking Velocimeter (MPTV) to measure the magnetophoretic mobility of particles up to 1,000 μm in diameter. Velocity measurements are performed in a well-characterized uniform magnetic energy gradient using video imaging followed by analysis of the video images with a computer algorithm that produces a histogram of absolute mobilities. MPTV was validated using magnetic agarose beads serving as islet surrogates and subjecting them to QMS. Mobility distributions of labeled porcine islets indicated that magnetized islets have sufficient mobility to be captured by the proposed sorting method, with this result confirmed in test isolations of magnetized islets.     

The use of regenerable, affinity ligand-based surfaces for immunosensor applications.

The regeneration of antibody-binding surfaces is of major importance for re-usable sensor formats such as required for direct 'real-time' biosensing technologies and is often difficult to achieve. Antibodies commonly bind the antigen with high avidity and may themselves be sensitive to regeneration conditions. The interaction of polyclonal anti-chlorpyriphos antibody with an immobilised chlorpyriphos-ovalbumin (chlor-oval) conjugate and the interaction of soluble recombinant CD4 with covalently immobilised anti-CD4 IgG are presented in order to highlight these difficulties. Affinity-capture is suggested as an alternative format as it facilitates surface regeneration, directed immobilisation and the attainment of interaction progress curves that conform to the ideal pseudo-first-order kinetic interaction model. Protein A, protein G and polyclonal anti-mouse Fe-coated surfaces were used to observe the interaction of captured anti-GST monoclonal antibody with glutathione-s-transferase (GST). It was shown that a protein A affinity-capture surface produced ideal interaction progress curves while both protein G and polyclonal anti-mouse Fe resulted in systemic deviations.     

Antibody-magnetite method for selective concentration of Giardia lamblia cysts from water samples.

An antibody-magnetite method was developed in order to selectively concentrate Giardia cysts from water samples. The indirect technique employed a mouse immunoglobulin G anti-Giardia antibody as a primary antibody and an anti-mouse immunoglobulin G antibody-coated magnetite particle as a secondary labeling reagent. The magnetically labeled cysts were then concentrated by high-gradient magnetic separation. Ninety percent of the seeded cysts were recovered from buffer when this method was employed. An average of 82% of the seeded cysts were recovered from water samples with various turbidities. Significantly higher cyst recoveries were obtained from water samples with turbidities below 600 nephelometric turbidity units.     

Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens.

BACKGROUND: In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. RESULTS: A second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab')(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not. CONCLUSIONS: Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.     

抗体滴定在血液细胞流式细胞术中的应用

流式细胞术（flow cytometry）可以实现高速、逐一的细胞定量分析和分选，是研究和诊断血液病的重要手段之一。但是由于不同实验所用细胞和实验条件不同，经常存在抗原阴性细胞非特异染色等问题。利用抗体滴定法，可通过计算、比较染色指数，得到使抗原阳性细胞群和阴性细胞群达到最佳分离效果的实验条件。为了优化血液细胞流式细胞术中荧光抗体染色的实验条件，以小鼠骨髓细胞为被标记细胞，选择利用非串联荧光染料FITC标记的大鼠抗小鼠CD11b抗体（FITC Rat Anti-Mouse CD11b）和串联荧光染料APC-eFluor780标记的大鼠抗小鼠CD11b抗体（APC-eFluor780 Rat Anti-Mouse CD11b）进行标记。通过计算不同浓度抗体标记小鼠骨髓细胞的染色指数进行抗体滴定，确定合适的抗体浓度区间，进而分析细胞数量、染色时间及固定步骤对抗体染色指数的影响，探究影响血液细胞抗体染色的关键因素。结果显示，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b的浓度分别在0.156~2.500 μg·mL-1和0.25~1.00 μg·mL-1范围内染色指数较高，但是超出这个范围的抗体浓度会使染色指数降低；抗体浓度、染色时间一定时，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b分别在细胞数量为1.56×105~5.00×106 cells·管-1和1.56×105~3.12×105 cells·管-1范围内染色指数较高，但是超出这个范围的细胞数量会使染色指数降低；抗体浓度、细胞数量一定时，对于FITC Rat Anti-Mouse CD11b，随着染色时间的延长，染色指数降低，而APC-eFluor780 Rat Anti-Mouse CD11b与之相反；通过比较固定前后染色指数的高低发现，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b在固定后染色指数均显著下降（P<0.01和P<0.05）。研究结果提供了一种通过抗体滴定优化流式分析血液细胞的方法，并指出在特定实验中根据抗体滴定结果选择合适的抗体浓度、细胞数量、染色时间和固定步骤对标记血液细胞进行流式检测的研究至关重要。     

Flow cytometric analysis of estrogen, progesterone receptor expression and DNA content in formalin-fixed, paraffin-embedded human breast tumors.

Flow cytometric analysis of estrogen (ER) and progesterone (PgR) receptor expression in archival human breast tumors is relatively difficult. We have used enzyme digestion and microwave antigen retrieval procedures for multiparametric flow cytometric analysis of ER and PgR expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded primary breast tumors. Deparaffinized rehydrated tissue sections treated with pepsin were subjected to microwave irradiation for unmasking of ER and PgR antigenic sites. Biotinylated ER antibody and streptavidin-fluorescein isothiocyanate (FITC) were used for ER labeling and PgR antibody with phycoerythrin labeled goat anti-mouse antibody was used for PgR labeling. Counter staining with propidium iodide-RNase was used for determination of cellular DNA content. Our results show that enzyme digestion and microwave treatment of formalin-fixed, paraffin-embedded breast tumors can be successfully used for the multiparametric analysis of nuclear hormone receptor expression and DNA content by flow cytometry.     

Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo

A mAb, 7G6, that binds to mouse CR1 and CR2 and down-modulates their expression on splenic B cells in vivo, was used to determine whether a decrease in CR1 and CR2 expression affects antibody responses to different T-dependent and T-independent Ag. Injection of mice with the mAb 7G6 prior to immunization with FITC haptenated Salmonella typhimurium (SH5771), Salmonella montevideo (SH5770), SRBC, or Ficoll dramatically decreased subsequent antibody responses to FITC. Although both IgM and IgG primary antibody responses were affected similarly, the antibody levels were most inhibited during early phases of the response. In contrast, down-modulation of the CR did not affect memory antibody responses, because injection of mice with 7G6 before a second immunization with FITC-SH5771 had no effect on subsequent anti-FITC antibody production. Moreover, polyclonal in vivo activation of the mouse immune system by anti-mouse IgD antibodies was not affected by previous administration of 7G6, because anti-IgD-induced increases in Ia expression and serum IgG1 levels were not affected. Taken together, these observations suggest that CR1 and CR2 may play an important role in enhancing primary antibody responses to many T-dependent and T-independent Ag and may contribute to a host's response to naturally occurring antigens such as bacteria.     

免疫磁珠分选降低分化体系中胚胎干细胞的比例

研究目的:采用免疫磁珠分选系统（magnetic activated cell sorting, MACS）分离去除小鼠胚胎干细胞（murine embryonic stem cells, mES）向神经细胞分化时培养体系中的ES细胞,即对分化细胞进行纯化,以期减少移植致瘤性。方法：诱导mES细胞向神经细胞分化,取分化第四期的细胞,胰酶消化制成单细胞悬液,用mES特异性表面抗原SSEA-1（special stage embryonic antigen-1）单抗标记,间接免疫磁珠分选系统分离去除SSEA-1阳性细胞,流式细胞仪检测分选前后细胞中mES细胞的比例,台盼蓝染色检测分选前后细胞存活率。结果：经MACS分选后的阴性细胞中的SSEA-1阳性率可以由分选前的（7.19±1.36）%下降到（1.34±0.80）%,结果具有显著性差异;分选后的细胞存活率仍为92%左右,与分选前存活率无明显变化。结论 用SSEA-1作为表面标志,用MACS方法能有效地去除胚胎干细胞分化细胞中残存的胚胎干细胞,得到高纯度的分化细胞,并且细胞存活率不受影响,为下一步进行移植实验奠定基础。