|
|||
|
Flow cytometry can also be utilized for purification of rare cell population by fluorescene-activated cell sorting (FACS). In a fluorescence-activated cell sorter, the fluorescence emitted by a labeled antibody bound to a cell marker of interest triggers a switch that sorts this cell into one or the other target reservoir. Modern cell cell sorters can sort as many as 300,000 cells per minute. FACS is utilized extensively in biomedical research as well as in experimental therapies such as bone marrow transplant, where harmful cell population can be sorted out from the donor marrow in order to minimize the risk of graft versus host disease. |
|||
 |
|||
Since its inception Antibody Solutions has been developing innovative flow cytometry applications and generated many antibody reagents optimized for flow cytometry. For example, we have pioneered intracellular cytokine staining of mixed cell populations with subsequent flow cytometric analysis. This technique can yield rapid and specific information on the cytokine production properties of individual cells within a mixed population of cells. The time from activating the cells to interpreting the data can be as short as 5 hours. Being able to look at individual cells and identifying their phenotype as well as their cytokine profile has tremendous benefits. Intracellular cytokine staining circumvents the need to isolate populations of cells by physical means such as panning methods, columns, density gradients, etc., which rarely yield populations of cells of sufficient purity that allow for easy data interpretation. The data presented above were generated by intracellular staining of peripheral blood mononuclear cells with our carboxyfluorescin-conjugated monoclonal antibody AS1-F to human tumor necrosis factor alpha (TNFa). This cytokine is secreted from many leukocytes in response to stimulation with bacterial lipopolysaccharide (LPS). In the left panel PBMC are gated into |
| Antibody Solutions has recently acquired a robotic Guava PCA-96 flow cytometer that can automatically analyze several stacks of 96-well plates for antibodies that bind to cell-surface receptors of interest. Conditioned media from hybridoma clones are screened for binding to cells expressing the antigen of interest, and positive samples are identified automatically based on the fluorescence of the gated cell population. This method is advantageous over cell-based ELISA, in that the signal to noise ratios tend to be much higher, and cell preparations do not have to be homogenous because the population of interest can be gated by a population-specific marker. Using this instrument, we are able to screen about 2000 samples per shift. View larger image of our robotic cytometer! |  |
Click on image to open larger cartoon |
Flow cytometry is limited to detection of cell surface or intracellular antigens. In order to overcome this limitation and measure secreted proteins such as antibody secreted from hybridoma cells, John S. Kenney (President and Research Director of Antibody Solutions) and colleagues developed an innovative flow cytometry application known as Secretion Capture and Reporter Web (SCRW), (Kenney et al., 1995; Gray et al., 1995). As illustrated on the left, secreting cells such as hybridoma are encapsulated in agarose microdroplets which have been derivatized to create a fluorescent antigen-specific sandwich assay. Flow cytometry can now be applied to these droplets to identify and sort productive cells from a heterogeneous population. Using agarase, the cells can be recovered from the microdroplets and clonally expanded after selection.
Antibody Solutions offers a wealth of expertise in developing flow cytometry applications and antibody reagents for these applications. Please contact us with your specific application needs. |
|
Antibody Solutions Tel: 888-843-1069 Fax: 650-938-4390 E-mail:  |
|
Webpage Design