Primary Moflo operator: Leslie Armstrong-Lea
Faculty Consultant: Anne Avery
Many possibilities exist for flow cytometric analysis or individual cell sorting, including, but not limited to:
- Immunofluorescent measurements (surface and intracellular markers, single and multi-parameter) for antigen and antibody distributions (up to 9 colors with 3 lasers)
- Determination of cell viability using Propidium Iodide, or PI with Sybr 14, and Dead/Live cell gating
- Expression of Green Fluorescence Protein (GFP), or ds-Red
- Detection of apoptosis with TUNEL or Annexin-V
- Multicolor identification or sorting of bacteria, parasites, neurons, sperm, or RBC’s
- Quantitative analysis of the number of molecules of a particular antigen found on the surface of a cell (MESF)
- Cell division studies using antibodies to bromodeoxyuridine (BrdU)
- DNA and cell cycle analysis, DNA Index for aneuploidy
- Mutant isolation
- Plant cell studies
- Virus infected cells
- Sterile sorting into multi-well plates, trays, or onto individual microscope slides with automated cloning device
- One, two, three or four way sterile tube sorting
Cell Sorting (top)
Cell sorting involves physically separating (purifying) a specific cell population from the remaining cells or particles in the suspension for the purpose of performing further experimental procedures on these purified cells. Sterile sorted cells can be cultured to expand the population. Cells can be re-stained with other fluorescent dyes to be re-analyzed, processed for RNA studies, or other experiments to be done to characterize the purified population.
In most cell sorters, cells are passed in a stream of fluid through the flow cell; they pass through a laser beam and are analyzed in the same way as in standard flow cytometry. The sample stream is broken into droplets at a fixed break off point. If a cell of interest passes through the laser beam, it is identified, and when it reaches the droplet of the break off point, an electric charge (positive or negative) is applied to the stream. As the droplet leaves the stream it passes through deflection plates carrying a high voltage and the droplet will be attracted to one of these plates, depending on the charge it was given. Uncharged droplets pass through to the waste, and deflected droplets are collected in tubes. In this way one, two, three or four different populations of cells can be sorted from the one sample. Cells can also be sorted using a cloning attachment, which has the capability of sorting single cells (or any specified number of cells) into 96 well plates (or any other size/number of wells), or directly on to a microscope slide.
There are a number of factors that determine the length of the cell sort, including cell type, cell size, cell concentration, sticky or clumpy cells, sort mode (purity or enrichment), and frequency (%) of the target cells.
Sorting speed is generally around 10,000 - 12,000 cells per second using the 100 micron flow cell tip for larger or sticky cells, and around 25,000 cells/second for smaller, well-suspended cells using the 70 micron flow cell tip.
Expected yields are usually 40% of the ideal yield on average. Highly purified cells may be less due to the number of aborted droplets. Higher yields are possible with less purity or enrichment.
Any BSL 2 material, including tissue from healthy human donors, is sorted with the aerosol evacuation unit running, and this adds some time to the sort. The sorting chamber must be evacuated before removing the sorted cells.
The purity of each sorted population will be tested after each sort by re-running the sorted population. . The purity of each sort can be compromised by several factors
- Unwanted cells (or platelets/debris) may be aggregating with cells of interest. These unwanted particles can separate after the sort, resulting in the contamination of a number of unwanted particles
- Loss of viability after the sort may result in cells exhibiting different light scatter properties from those of the original cells.
- The capping of cell surface markers after the sort will often result in sorted cells that are somewhat less fluorescent than the original selected population.
- Dead cells can non-specifically bind antibody and thus can appear to the flow cytometer as positive events. The flow cytometer can discriminate most dead cells, but cells that have recently died or are not healthy can lead to false positive results.
- The cells of interest are not completely resolved from the unwanted cells (overlapping populations)
Example: Post Sort Purity Check of FITC Positive B-Cells
Flow Cytometry (top)
Flow cytometry is the characterization of single cells as a fluid stream of cells is passed through a laser beam at high speed (thousands of cells/second). Flow cytometry uses a laser-based instrument to analyze and sort cells or particles based on their light scattering properties and their pattern of fluorescence emission. Not only is the analysis fast, it yields information that is unattainable by other methods. As each cell flows past a focused laser beam (or beams) of appropriate wavelength(s), light is scattered and detected in both the forward and side (90 degree) parameters. If the cell contains any molecules that fluoresce from the wavelength of the laser light, fluorescence is emitted; this allows looking at the cells with the property associated with that particular fluorescence. This information is used to determine the characteristics of those cells. Flow cytometry provides data on large numbers of individual cells, and is very fast- thousands of cells per second can be analyzed and/or sorted (purified). The information gathered from each cell is stored. To allow for multiple different biological or biochemical properties of the cells to be determined at one time, the cells can be stained with different fluorescent dyes which bind specifically to the cellular component of interest. Forward scatter gives an indication of a cell's relative size and side scatter an indication of its texture (granularity). Fluorescence depends on the binding of the different fluorescent dyes for the property of interest.
Examples: (top) (bottom)