What is the top flow cytometer? The easy answer is the flow cytometer that matches your needs and fits within your budget. However, before running off to spend cash, consider the following. What are the current needs of the users? Evaluating the user’s needs will help define the parameters needed for the flow cytometer. This will include what excitation sources should be available, how many detectors are needed, including specialized detectors (small particle detectors), as the need for automatic sampling. All these factors will in weigh in on the What are the projected future needs of the users? This is…
As with all complex technology there are many different levels of education that users should avail themselves of. Basic instrument operation This level of education is akin to learning how to drive a car. At this level, the focus will be on how to put the sample on the instrument, how to adjust voltage and collect data. Specific policies of the facility should also be covered. Advanced instrument operation At this level of education, the focus should be on advanced techniques on the instrument. This would cover basic troubleshooting – identifying problems and how to solve them. Advanced training should…
Cell proliferation can readily be measured by flow cytometry. Depending on the research question, there are several different techniques that can be used. Cell counting experiments This relatively straightforward experiment where the investigator uses one of several counting techniques to see if there is an increase in the populations. This can be done using a microscope and a hemacytometer, a flow cytometer with counting beads, image based tools (like the T-20, the Countess, or the Cellometer), or coulter counter type instruments (including the Coulter Counter the Scepter and the Casy counter) Cell cycle experiments One of the earliest techniques developed…
Flow cytometry is a powerful tool for asking and answering questions at the whole cell level. The first step in any flow cytometry experiment is to define the hypothesis or biological question that is to be answered. This helps ensure that flow is the correct technique for answering the question. One major determination that needs to made is will the cells be purified (via sorting) or is the experiment just analytical. The second step in any flow cytometry experiment is to identify the reagents needed for the experiment. In immunophenotyping experiments, this involved knowing what subpopulations that need to be…
Flow cytometry can be an intimidating tool for the new cytometrist. There are many sources that one can turn to for training and education. Local Shared Resource Lab Manager If you institute has a shared resource lab (a ‘core facility’), look in what training they provide. This is your first line of training experience. Here you can learn about the policies of the facility, how to operate the machine and how to design or analyze your experiment. These SRL managers and staff have extensive experience with flow cytometry and can help guide you through the steps to get good, high…
Cell sorting remains the best tool to isolate and purify cellular populations that can be phenotypically defined. This is especially true for rare-event detection and purification. Successful rare event detection and purification requires some attention to ensure the best yield and purity. 1) Watch the instrument to ensure success a) Keep the core stream tight – the tighter the core stream (low differential pressure), the tighter the CV on resulting data, therefore the easier it is to determine best placement of gates for sorting. Tight core streams also reduce coincident events (two cells passing through the intercept at the same…
Mass Cytometry, commercialized by the company DVS Sciences, in the instrument called the CyTOF is a newly emerging technology in the field of flow cytometry. This technology replaces traditional fluorescent-labeled antibodies with highly purified, stable isotopes with very well characterized mass values. This extends the power of flow cytometry from 14-18 fluorochromes to over 40+ simultaneous parameters. The advantage of mass cytometer include: ● Increased number of parameters measured ● No equivalent autofluorescence – cells do not contain stable lanthanide ions ● Minimal ‘compensation’, mostly due to oxidation of some metals, which is predictable Some limitations of the mass cytometer…
Cell sorting can be a scary proposition. A precious sample is introduced into a machine that pressurizes the cells to 70 PSI, moves them past one or more lasers, vibrates the stream at 90 kHz before decelerating the cells to atmospheric pressure before they hit an aqueous surface. Many cells survive this journey. But some do not. Here are 10 things smart scientists do to improve their cell recovery: 1. They pre-coat their catch tubes. A smart way to improve your cell recovery is to incubate your plastic tubes with a buffer solution containing protein. This will help reduce/eliminate the…
Cell Sorting is the process of isolating cells after the identification of the cells using the principles of flow cytometry. The upstream components of the cell sorter are common to all flow cytometers. The difference comes in what is done with the cells after they have been interrogated and identified. The stream is vibrated to generate thousands of individual droplets (as many as 90,000 or more), a fraction of which contain a cell. Those droplets that contain a cell of interest can beelectrically charged, as as the pass into an electric field, are deflected to the final receptacle, as shown…
A variation on biexponential scaling similar to logicle scaling. The biexonential scale is a combination of linear and log scaling on a single axis using an arcsine function as its backbone. Biexponential scales are more generally referred to as hybrid scales and include other variations like lin/log or log with negative. More information on Hyperlog scaling can be found here: Bagwell, CB. (2005). Hyperlog-a flexible log-like transform for negative, zero, and positive valued data. Cytometry. 64: 34-42.
Cell cycle analysis by flow cytometry uses a DNA binding dye, such as propidium iodide (PI), 7- aminoactinomycin D (7-AAD) or 4’,6-diamidino-2phenylindole (DAPI), to determine the cell cycle state of a cell population. The Gap1 (G1) phase of an eukaryotic cell is defined as having 2C DNA. The synthesis (or S) phase is where the DNA is synthesized going from 2C->4C. Cells then spend some time in the Gape 2 (G2) phase before completing mitosis and the whole cycle starts over again. Since the cells in G0/G1 and G2/M have defined amounts of DNA, with the S phase having an…
The question always arises as to what is the top cell sorter on the market. This question is a difficult one to generalize because there are several considerations that need to be made in choosing a cell sorter. What are the sorting needs of the investigators? If all the investigators do is sort GFP positive cells, then a simple sorter like the Bio-Rad S3 fits the bill. On the other hand, of the investigators need to do 4-12 color experiments with four way sorting, the choice becomes more muddled. Who will operate the instrument? Is the instrument going to be…