Purification of transiently transfected cells by magnetic affinity cell sorting

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.     

Bialaphos selection of stable transformants from maize cell culture

Summary Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for -glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.     

Isolation of a population of transiently transfected quiescent and senescent cells by magnetic affinity cell sorting.

Rous sarcoma virus (RSV) and cytomegalovirus (CMV) promoters were tested for activity in proliferating and nonproliferating (quiescent or senescent) human embryo fibroblasts. These promoters were cloned upstream of the coding sequence for the Tac subunit of the interleukin 2 receptor, and activity was calculated from the fraction of Tac antigen positive cells detected in a coupled transient transfection/magnetic affinity cell sorting assay. Differences in promoter activities are substantial in quiescent cells: the efficiency of the RSV promoter is no greater than background whereas the CMV promoter is equally active in serum concentrations ranging from 0.5 to 20%. While both promoters are functional in growing cells (WI-38 and HeLa), the CMV promoter exhibits twofold greater activity. Surprisingly, in senescent cells both promoters exhibit the same degree of activity.     

Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines.

The production of Green Fluorescent Protein in recombinant NIH3T3 mouse fibroblast cells was used as a model to determine the optimal conditions for the rapid isolation of high-producing cell lines with a fluorescence-activated cell sorter. "Bulk sorting", that is, sorting of a large number of positive cells, did not result in a stable, high-producing cell line due to overgrowth of high-producing cells by low- or nonproducing cells. The production kinetics and expression of GFP during batch culture was found to differ between NIH3T3 cells and HepG2 hepatoma cells, even though the same plasmid was used for transfection. The kinetics of product formation need therefore to be determined from case to case to select the optimal timepoint for analysis and sorting. Subcloning of sorted cells into microtiter plates only resulted in high-producing subclones when 1 or 2 cells were seeded per well. Higher seeding rates again resulted in overgrowth of low- or nonproducers. By subcloning, two high-producing cells lines could be isolated. They had a 10- and 15-fold higher fluorescent signal compared to the negative control. While one of these subclones started to decrease it's GFP expression after 2 months, the other clone stably expressed GFP for 4 months.     

Evaluation of selectable markers for obtaining stable transformants in the gramineae

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10⁻⁵ to 2 × 10⁻⁶) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.     

Coral cell separation and isolation by fluorescence-activated cell sorting (FACS)

Background

Generalized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians.

Methods

Using fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay.

Results

We found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker.

Conclusions

In this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies.

     

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.     

Individual cell sorting.

Current cell sorting machines do not preserve the individual identity of processed cells; after analysis, the cells are assigned to a subpopulation where they are pooled with other similar cells. This paper reports progress on a system that sorts cells individually to precise locations on a microscope slide and preserves them for further observation with a light microscope while recording flow measurement data for each cell. Various electronic and mechanical modifications to an existing sorting machine are described that increase drop placement accuracy and permit individual cell sorting.     

An instrument for sorting of magnetic microparticles in a magnetic field gradient.

BACKGROUND: The goal of our bioassay technique is to demonstrate high throughput, highly parallel, and high sensitivity quantitative molecular analysis that will expand current biomedical research capabilities. To this end, we have built and characterized a magnetophoresis instrument using a flow chamber in a magnetic field gradient to sort magnetic microparticles by their magnetic moment for eventual use as biological labels. METHODS: The flow chamber consists of a sample inlet, differential sheath streams, and eight outlets for collecting the microparticles after they have traversed the chamber. Magnetic microparticles are injected into the flow chamber that is positioned in a linear magnetic field gradient. The trajectory for each microparticle is determined by its total magnetic moment and size. The resulting populations of monodispersed magnetic microparticles in the different outlet bins are sorted by their magnetic moment; with the highest magnetic moments being deflected the furthest. RESULTS: We have characterized the system for sorting both superparamagnetic and ferromagnetic microparticles with approximate diameters of 8 microm and 4.0-4.9 microm, respectively. To characterize the instrument, we used microparticles with a known size distribution and varied the transit time through the chamber. This is equivalent to varying the magnetic moment, while allowing us to hold the particle properties constant from run-to-run. We demonstrated the ability to reproducibly change the distribution of the particles in the collection bins by varying transit time in good agreement with theory. We identified hydrodynamic instabilities responsible for causing dispersion in the flow. Improvements to the flow chamber hydrodynamics such as reducing the aspect ratio between the sample inlet and the chamber depth and stabilizing the sheath flow resulted in narrow sorting distributions. We measured a sorting reproducibility (percentage of particles returning to their original bin upon resorting individual populations) of 84-89%. CONCLUSIONS: We have developed a simple magnetophoresis system for reproducibly sorting magnetic microparticles. This technique will permit the use of microparticles with a wide range of magnetic moments to create a wide range of magnetic labels. Careful consideration of system design and operational parameters enables reliable and reproducible sorting of microparticles with varying size and magnetic content.     

Preparation of a stable folate-sepharose complex for affinity chromatography.

A stable folic acid affinity gel has been developed for the purification of nanograms of protein that bind folic acid or its derivatives. The affinity gel was prepared by first coupling folic acid covalently to bovine serum albumin, followed by covalent coupling of the albumin to p-benzoquinone-activated Sepharose. After the albumin-folic acid complex was formed, it was treated with charcoal to remove ionically bound folate which would otherwise elute from the gel and decrease the recovery of the binding protein. The p-benzoquinone activation resulted in a more stable binding of the albumin to the Sepharose.     

An evaluation of lectin-mediated magnetic bead cell sorting for the targeted separation of enteric bacteria

Lectin-labelled magnetic beads were assessed and compared with antisera as an alternative approach for the targeted separation and isolation of enteric bacteria. Of the 16 lectins tested against a range of bacterial species, concanavalin A (conA) showed the greatest potential. Agglutination of bacterial cells in suspension using conA and methods for effective labelling of the magnetic beads with the lectin were optimized. ConA-labelled magnetic beads were compared with antibody-labelled beads for recovery of bacterial cells from pure or mixed laboratory cultures and from natural populations in river water. Recovered cell populations were free from environmental impurities and a high percentage of the culturable cells was extracted. Specific cell recovery was found to be variable, but the use of lectins offers some promise as an alternative cell discriminator.     

Rapid isolation of single malaria parasite-infected red blood cells by cell sorting

Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum-infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.     

Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting

We have used highly specific, directly fluorescein-conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10-5 to 10-6. We were able to isolate the variants by first sorting for an approximate 1000-fold enrichment of the desired immunoglobulin-producing cells, growing these cells for five to nine days, followed by a second 1000-fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3-5 weeks after the original selection for secretion of dansyl-binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, "red" fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting.     

Improvement of phytoplankton culture isolation using single cell sorting by flow cytometry

Flow cytometry provides a tool to physically sort single algal cells in order to obtain clonal cultures. During sorting, cells are submitted to physical stress factors such as high fluidic pressure, exposure to the laser beam, electrostatic charges, deflection through high voltage fields, and collisions with container surfaces. All of these can damage the cells of interest and success rates for initiation of cultures from flow‐sorted cells are generally very low. We found that the addition of bovine serum albumin in the culture medium into which cells were sorted drastically improved the success of initiation of pico‐ and nano‐eukaryotic phytoplankton strains. Adding a mixture of antibiotics (Penicillin, Neomycin, Streptomycin) to the medium in order to slow down bacterial growth further improved culture development. This approach was successfully used to isolate taxonomically diverse strains, including novel taxa, from a fresh sample obtained in the English Channel and from enrichment cultures established during an Atlantic meridional transect cruise. We anticipate that these improvements will be useful to clone or purify existing cultures and to isolate novel cultures from oceanic samples.     

Boundaries in gravitational and magnetic activation of cells for sorting.

Standard deviations in the distribution of radii of cells and particles are considered to arrive at realistic limits in the use of gravitational and magnetic activation of cells for sorting. Using a specific fractionation design, it is shown that the radius of particles (or cells) may be fractionated down to a precision of +/- 0.76%. Although higher precisions could be obtained with other designs, the number of particles available per fraction is inversely proportional to the precision desired. Thus, one would prefer to keep the precision as moderate as permissible by the experiments.     

Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display

We have investigated a staphylococcal surface display system for its potential future use as a protein library display system in combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1:1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections.     

Magnetic biospecific affinity adsorbents for lysozyme isolation

Summary Magnetic biospecific affinity adsorbents for lysozyme isolation have been prepared. They were obtained by incorporation of fine magnetite particles into the structure of chitin, agar or agarose. Hen egg white lysozyme was obtained in 90% purity in one step.