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.     

Detection and separation of the submetacentric marker chromosome of the WALKER (W-256) carcinoma using flow cytometry and sorting

Summary The chromosomes of WALKER (W-256) carcinoma cells have been separated into different DNA subclasses using DAPI for quantitative DNA staining and laser flow cytometry. The submetacentric marker chromosome could be isolated and its DNA content was determined to be 1.3 pg. One microgram marker DNA was obtained after separation of about 750 000 marker chromosomes by means of electronic flow sorting. The chromosomal composition of sorted fractions was analyzed by microscopy following banding of sorted chromosomes. The average morphological purity obtained was about 83%.     

Detection and separation of the submetacentric marker chromosome of the WALKER (W-256) carcinoma using flow cytometry and sorting

The chromosomes of WALKER (W-256) carcinoma cells have been separated into different DNA subclasses using DAPI for quantitative DNA staining and laser flow cytometry. The submetacentric marker chromosome could be isolated and its DNA content was determined to be 1.3 pg. One microgram marker DNA was obtained after separation of about 750 000 marker chromosomes by means of electronic flow sorting. The chromosomal composition of sorted fractions was analyzed by microscopy following banding of sorted chromosomes. The average morphological purity obtained was about 83%.     

免疫磁珠法分离鼻咽癌基质的T淋巴细胞

肿瘤内环境与肿瘤的发生密切相关.肿瘤细胞周围的组织在癌变发生时不会是一个沉默的旁观者,可能在肿瘤的发生和发展中扮演十分重要的角色.本研究分别采用不同的磁珠分选技术分离T淋巴细胞.采用CK LMP1,CD105和成纤维细胞表面蛋白,结合全血总T细胞试剂盒,间接法分离鼻咽癌基质的T淋巴细胞;采用CD3直接磁分选法分离鼻咽癌基质的T淋巴细胞,然后用免疫组化法鉴定分选的效果和细胞的质量.结果表明,免疫组化显示间接磁分选法分离出来的T淋巴细胞不能完全去除肿瘤细胞,RNA的质量不佳;而直接磁分选分离出来的T淋巴细胞为纯净的T淋巴细胞,而且RNA的质量良好.提示直接磁分选技术是分离鼻咽癌基质T淋巴细胞的首选方法.     

Hyphenation of sedimentation field flow fractionation with flow cytometry

Interest in the development of field flow fractionation (FFF) systems for cell sorting recently increased with the possibility of collecting and characterizing viable cellular materials. There are various tools for the analysis of cell characteristics, but the reference is small- and large-angle light scattering often coupled with fluorimetric measurements. The well-known flow cytometry (FC) cell analysis techniques can be associated with FFF leading to the possibility of collecting information provided by a remarkable separation technique for micron-sized particles (cells) operating in the steric-hyperlayer elution mode with multiparametric detection provided by flow cytometry. Moreover FFF derived cell characteristics can be correlated with FC characteristics to describe in a unique way the nature of the eluted materials. Experimental demonstrations are described herein using nucleated cells (HL-60 cell lineage) and human red blood cells (HRBC).     

Fluorescence-activated cell sorting of specific affibody-displaying staphylococci

Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.     

Integrating cytomics and proteomics

Systems biology along with what is now classified as cytomics provides an excellent opportunity for cytometry to become integrated into studies where identification of functional proteins in complex cellular mixtures is desired. The combination of cell sorting with rapid protein-profiling platforms offers an automated and rapid technique for greater clarity, accuracy, and efficiency in identification of protein expression differences in mixed cell populations. The integration of cell sorting to purify cell populations opens up a new area for proteomic analysis. This article outlines an approach in which well defined cell analysis and separation tools are integrated into the proteomic programs within a core laboratory. In addition we introduce the concepts of flow cytometry sorting to demonstrate the importance of being able to use flow cytometry as a cell separation technology to identify and collect purified cell populations. Data demonstrating the speed and versatility of this combination of flow cytometry-based cell separation and protein separation and subsequent analysis, examples of protein maps from purified sorted cells, and an analysis of the overall procedure will be shown. It is clear that the power of cell sorting to separate heterogeneous populations of cells using specific phenotypic characteristics increases the power of rapid automated protein separation technologies.     

Applications of flow cytometry sorting in the pharmaceutical industry: A review

The article reviews applications of flow cytometry sorting in manufacturing of pharmaceuticals. Flow cytometry sorting is an extremely powerful tool for monitoring, screening and separating single cells based on any property that can be measured by flow cytometry. Different applications of flow cytometry sorting are classified into groups and discussed in separate sections as follows: (a) isolation of cell types, (b) high throughput screening, (c) cell surface display, (d) droplet fluorescent-activated cell sorting (FACS). Future opportunities are identified including: (a) sorting of particular fractions of the cell population based on a property of interest for generating inoculum that will result in improved outcomes of cell cultures and (b) the use of population balance models in combination with FACS to design and optimize cell cultures.     

A flow cytometric assay for screening improved heterologous protein secretion in yeast

Selection of highly productive hosts for protein expression is a significant component of bioprocess design. As an alternative to traditional plate, halo, and suppression-based screens, we describe a high-throughput, flow cytometric assay, the Cell Surface Secretion Assay (CeSSA), that can be used to select for improved heterologous protein secretion from a population of S. cerevisiae mutants. A ligand is covalently attached to the cell surface via a PEG linker, and as cells secrete a protein that binds the tethered ligand, the protein is captured on the surface where it can be labeled and the cells sorted using flow cytometry. This report describes three different protein/ligand interactions that have been demonstrated with this system. Single-pass sorting enrichments from 23- to 54-fold have been validated in the separation of a 3-fold higher secretor from a background population of wild-type secretors making this system applicable to large library screening (10(8) clones). A mathematical model was developed to improve the parameters of the assay further. The model was validated with time course data and predicts an optimal screening window. The model also predicts a 60-fold enrichment rate for the validation experiment described above. With the development of this selection system, limitations presented by traditional, particularly plate-based, secretion assays can be overcome so that a larger search space can be examined under conditions closer to the growth physiology experienced by cells in fermentors.     

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。     

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.     

Separation of sperm and vaginal cells based on ploidy, MHC class I-, CD45-, and cytokeratin expression for enhancement of DNA typing after sexual assault.

BACKGROUND: Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility (MHC) class I, CD45 and cytokeratin expression. METHODS: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. RESULTS: The sorting procedure was superior to the preferential lysis method within all tested dilutions. One documented case of rape was examined with both procedures and only after cell sorting with flow cytometry was the male DNA identified. CONCLUSIONS: We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there is only a few sperm detectable after rape.     

Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci

Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.     

An improved cell separation technique for marine subsurface sediments: applications for high‐throughput analysis using flow cytometry and cell sorting

Development of an improved technique for separating microbial cells from marine sediments and standardization of a high‐throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ～ 50% of cells were recovered from deep samples (100–365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 10⁴–10⁸ cells cm^?3. We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high‐throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single‐cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere.     

High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening

HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.     

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.     

Purification of pluripotent embryonic stem cells using dielectrophoresis and a flow control system

Pluripotent stem cells (PSCs) such as embryonic stem cells and induced PSCs can differentiate into all somatic cell types such as cardiomyocytes, nerve cells, and chondrocytes. However, PSCs can easily lose their pluripotency if the culture process is disturbed. Therefore, cell sorting methods for purifying PSCs with pluripotency are important for the establishment and expansion of PSCs. In this study, we focused on dielectrophoresis (DEP) to separate cells without fluorescent dyes or magnetic antibodies. The goal of this study was to establish a cell sorting method for the purification of PSCs based on their pluripotency using DEP and a flow control system. The dielectrophoretic properties of mouse embryonic stem cells (mESCs) with and without pluripotency were evaluated in detail, and mESCs exhibited varying frequency dependencies in the DEP response. Based on the variance in DEP properties, mixed cell suspensions of mESCs can be separated according to their pluripotency with an efficacy of approximately 90%.     

Microsystems for isolation and electrophysiological analysis of breast cancer cells from blood

This paper presents the development of a microsystem for separating suspended breast cancer cells in peripheral blood and for sorting them based on their electrophysiological characteristics. A continuous paramagnetic capture mode (PMC) magnetophoretic microseparator was utilized for the isolation of suspended breast cancer cells in peripheral blood based on the native magnetic properties of blood cells without any tagging such as with magnetic probes. A micro-electrical impedance spectroscopy (mu-EIS) system was used as a downstream cell analysis tool to extract the pathological characteristics from the breast cancer cells. The system was fabricated on silicon and glass substrates utilizing microfabrication and stereolithography technologies. The experimental results of the PMC microseparator show that 94.8% of the breast cancer cells could be continuously separated out from a spiked blood sample with a 0.2 T external magnetic flux. The electrical impedances of human breast cancer cell lines of different pathological stages (MCF-7, MDA-MB-231, and MDA-MB-435) were measured using mu-EIS and compared to those of normal human breast tissue cell line MCF-10A.     

Improvement of flow-cytometric detection of multidrug-resistant cells by cell-volume normalization of intracellular daunorubicin content

To improve the ability of flow cytometry to detect multidrug-resistant cells, we studied the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content. For P388 murine or HL-60 human leukemia cells exposed to DNR (1 micrograms/ml, 60 min), log intracellular DNR content varied in direct proportion to log cell volume measured by flow cytometry, with a correlation coefficient of .9. This relationship was confirmed by cell sorting based on intracellular DNR content with subsequent volume determination of the sorted cells. Normalization of intracellular DNR content for cell volume (thus obtaining intracellular DNR concentration) was accomplished by subtracting log cell volume from log intracellular DNR content for each cell. This resulted in a 34% decrease (range 23-58%) in standard deviation compared to DNR content measurements without volume normalization for all cell types tested. Following exposure to DNR (as above), intracellular DNR content of drug-sensitive P388 or HL-60 cells measured by flow cytometry was 12- and 8-fold greater than that of the multidrug-resistant sublines P388/ADR and HL-60/AR, respectively. However, because of the variance of intracellular DNR content, the predictive value of flow-cytometric determination of intracellular DNR content as a discriminant assay for detecting the frequency of drug-resistant cells in a mixed population was acceptable only when the frequency of resistant cells in the population exceeded 10%. In contrast, volume normalization of intracellular DNR content enhanced the ability of the flow-cytometric assay to discriminate resistant cells by 10-fold for P388 cells and 100-fold for HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)