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Flow Cytometry Training

Flow Cytometry Training

By: Tim Bushnell, PhD

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…

Sorting

Sorting

By: Tim Bushnell, PhD

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…

CyTOF

CyTOF

By: Tim Bushnell, PhD

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…

10 Things Smart Scientists Do Before Sorting Cells

10 Things Smart Scientists Do Before Sorting Cells

By: Tim Bushnell, PhD

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

Cell Sorting

By: Tim Bushnell, PhD

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…

Hyperlog Scaling

Hyperlog Scaling

By: Tim Bushnell, PhD

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

Cell Cycle Analysis

By: Tim Bushnell, PhD

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…

Top Cell Sorter

Top Cell Sorter

By: Tim Bushnell, PhD

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…

Critical Steps in DNA Cell Cycle Analysis

Critical Steps in DNA Cell Cycle Analysis

By: Tim Bushnell, PhD

DNA cell cycle analysis is a very powerful technique in flow cytometry. It is deceptively easy, but there are several critical things to remember to ensure successful analysis. Collect enough events. Cell cycle analysis involves fitting of the data using one of several mathematical models that describe the behavior of the data. These models make different assumptions about the S phase as well as the G1 and G2/M phases. To have enough data, one should collect 100 events for each channel between the beginning of the G1 peak and the end of the G2/M peak. Thus, if the G1 peak…

How To Perform A T-Test

How To Perform A T-Test

By: Tim Bushnell, PhD

With the ability to capture expression data at the single cell level through many thousands of cells in a short time, flow cytometry data is very numbers rich. The importance of those numbers and how to use them in hypothesis testing is critical to ensure the robustness of the analysis. After establishing the null hypothesis for the experiment, the type of statistical test, and the numbers necessary will become obvious. For example, if the null hypothesis states that the ‘treatment of B cells with thiotimoline does not change the expression of CD221B in normal patients.’ Based on this null hypothesis:…

Logicle Scaling

Logicle Scaling

By: Tim Bushnell, PhD

An implementation of biexponential scaling published by the Herzenberg lab at Stanford. The biexonential scale is a combination of linear and log scaling on a single axis using an arcsine function as its backbone. The “logicle” implementation of biexponential was implemented in many popular software packages like FACSDiva and FlowJo. Other types of biexponential scaling exist, including Hyperlog. Biexponential scales are more generally referred to as hybrid scales and include other variations like lin/log or log with negative. More information on logicle sclaing can be found here: Parks DR, Roederer M, Moore WA. (2006). A new “Logicle” display method avoids…

6 Flow Cytometry Gating Tips That Most Scientists Forget

6 Flow Cytometry Gating Tips That Most Scientists Forget

By: Tim Bushnell, PhD

After completing the perfect staining and cytometry run, the hard work begins – data analysis.  To properly identify the cells of interest, it is critical to pull together knowledge of the biology with the controls run in the experiment to properly place the regions of interest that will be dictate the final results.  Gating is an all-or-nothing data reduction process.  Cells inside the gate move to the next checkpoint, while cells outside the gate – even by a pixel, are excluded. 1.  Before beginning, know as much as you can about the populations of interest. While it may sound flip,…

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.