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Flow Cytometry Basics

Definition: Flow cytometry is a technique allowing for the examination of large numbers of single cells at high speed. The principle involved that cells can be passed within a sheath of solvent so that they pass a laser as individual units. The laser is employed to capture a scatter profile of the cells that gives information about the size and internal complexity of the cells and may also excite fluorescent probes that identify specific surface or internal structures.

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from: flowcytometry.med.ualberta.ca

Cytometers record information about each individual cell across a number of characteristics. To accomplish this, cells are titrated to run at a speed (measured by cells/second – often around 10,000 cells/sec) that is within the capacity of the machine to read. Physically, a constant flow of ‘sheath fluid’ is run across the detector’s path. The cell suspension runs as a separate stream within the sheath fluid.

 Data points can be represented very clearly as values for each characteristic measured, and may be listed as a series of numbers as the table below. Here, cells were ‘labeled’ with antibodies against three known proteins, Btk, CD3, and CD19. Each antibody also carried fluorophores that emit known wavelengths of light when excited by (a) laser(s) of specific wavelength(s). These antibodies are illustrated in the figure to the side. Each type of antibody binds to a specific ‘antigen’ and carries several fluorophores that have been chemically linked to them Screen Shot 2016-07-10 at 10.30.01 PM(illustrated by different colored stars). Alternatively, secondary reagents can be used to bind to the primary antibodies to allow more freedom of color choice or to amplify weaker signals.

Cells are labeled or ‘stained’ with these antibodies by incubating cells with the antibodies for a period of time, followed by washes to remove excess, unbound antibodies. Typically, all stains can be done together in a single incubation unless secondary antibodies are employed to amplify weak signals or adjust the colors used or intracellular staining is required (see below).

ID# FSC SSC Btk-FITC CD3-PE CD19-APC
1 412 183 41 6 58
2 374 192 4 745 9
3 299 201 3 4 8

If we measured data from each of the three cells above, this might be sufficient to illustrate the identity of each cell type without further analysis. However, if thousands of cells are measured for each condition in an experiment (done in triplicate), tables of numbers lose their value as effective illustrations of the data.

To account for this, scatter plots or density plots (similar to topographical maps) are regularly used to illustrate these larger datasets. Because it is only practical to present values in two dimensions at a time, plots are often drawn such that a population is identified in one plot and then those ‘gated’ cells are then redrawn in subsequent plots to illustrate values in new categories. Cells may also be examined for just one characteristic using a histogram.

Forward Scatter (FSC) and Side Scatter (SSC)

FSC and SSC are (very often) the primary measures of the physical properties of cells as they pass through the cytometer’s laser. FSC provides information about the size of the cell, while SSC provides information about the internal complexity of the cell. These data are presented for a sample dataset of white blood cells below. The more numerous Red Blood Cells (RBCs) and platelets have been eliminated prior to analysis.

Screen Shot 2016-07-10 at 10.30.33 PMThe cells illustrated in the FSC / SSC plot above fall into identifiable subsets of white blood cells based on their size and complexity. The gated cells are known as lymphocytes, which includes both B and T Cells. Gating is a way of selecting a group of cells to analyze further.

Screen Shot 2016-07-10 at 10.30.41 PMFluorescence

Here, the lymphocyte population is now distinguished by the presence of identifying surface proteins, CD19 (found on B Cells) and CD3 (found on T Cells). By plotting the fluorescence emitted by antibodies to these receptors, we can not only identify the two major populations but gate each of them for further analysis for another protein, the intercellular protein kinase, Btk.

Looking at the Btk expression requires a slightly different technique because this protein is located inside the cell. For antibodies to access to Btk, we have to punch holes in the cell that let antibodies permeate cells. This is done chemically after all surface staining is complete and cells are ‘fixed.’ Otherwise, the protocol is very similar to surface binding.

Screen Shot 2016-07-10 at 10.30.49 PMIn the last panel, both B Cells and T Cells (individually identified previously) are assessed for the presence of Btk and the results are represented as the number of events (cells) exhibiting high or low expression (illustrated below).

Here we can see that the B Cells express uniformly high levels of Btk, while T Cells express little or none. It would also be possible for us to see if only a subpopulation of either B or T cells expressed the kinase. In that case, we could gate expressers vs non-expressers to see if there are any other indications that these cells are different such as cell size or expression levels of the other receptors (CD19 or CD3).

It is possible to use staining to examine other features of the cells as well. For instance, if a treatment of cells might result in cell division, this can be tracked by using a non-toxic dye which is added to cells prior to treatment and then assessed afterward (typically 3-5 days). Because the dye is added only once, cells that divide will each take only half the quantity of the original dye. It is possible to distinguish up to 4-5 divisions clearly.

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CellTrace is as a ThermoFisher product, this graphic was taken from the product literature

Data from these proliferation assays is often viewed in histograms to see the proportion of cells at each division, or with another label to see if the dividing cells up- or down-regulate certain receptors. It is also common to use a vitality dye that would demonstrate if cells that don’t divide die, vice versa, or exhibit some other pattern.  The cells illustrated below are CD4 T Cells that were induced to divide by a ‘mitogen,’ possibly IL-2. The histograms depict cells in each generation, where the generation farthest to the right is the parent generation (i.e. undivided cells – this would be confirmed by a control population grown without mitogen). The next peak to the left represents cells that have divided once, the next represents cells that have divided twice, and so on. In quantitating the number of cells that have divided, it is important to consider that ONE parental cell is responsible for TWO cells that have divided once or FOUR cells that have divided twice, etc. (Note that the CellTrace dye is plotted along a log axis)

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Also from the CellTrace product literature

The Scatter plot illustrates the same cells, also plotted by cell division on the X- axis, however, this time the Y-axis separates cells according to their expression of CD4. These data show that the most actively divided cells are divided between CD4 expressors and non-expressers. We can also see that CD4 expression spikes in expressors upon division.

 

Definitions:

Fixing – chemically attaching antibodies to their targets in a reaction that kills the cells. This is required for longer term storage of cells and if further processes such as intracellular staining must be done.

Gating – Drawing a limit around a group of cells or an area where cells might appear for further analysis and/ or quantitation. Gating will always result in a calculation of the percentage of the total cells that are included within the gate

Labeling / Staining – to add fluorescent reagents to cells that will bind to specific elements.

Mitogen – a substance that induced cell division.

Washing – to repeatedly add a solvent to cells, spin to pellet the cells and remove unbound materials with the solvent.

 
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Posted by on July 10, 2016 in Uncategorized

 

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Another Puzzle – about the interaction of host and pathogen

Ok, I realize that some of the past puzzles I’ve made either had errors or were simply too obscure in their clues (again, I’m a crossword novice). I tried to be a little more clear with this puzzle and also did my best to force as many clues into actually ‘crossing’ as possible.

Let me know what you think and if you suspect any errors. The topic is: the host-pathogen interaction, our first chapter on immunology.

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Clues

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Posted by on April 15, 2014 in Uncategorized

 

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Son of HeLa to speak at JCCC Feb 21

Last week in Microbiology, we mentioned the use of HeLa cells in the context of informed consent. This week, I found out that the son of Henrietta Lacks is appearing to speak at JCCC this month.

From the JCCC website:

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What is it like to know that cells from your mother were taken without her consent to create a global strain of cells used the world over?

David “Sonny” Lacks will answer that question and more when he visits Johnson County Community College.

In an Actor’s Studio-like conversation at 11 a.m. Thursday, Feb. 21, in Polsky Theater in the Carlsen Center, he’ll discuss his mother, Henrietta Lacks, a poor tobacco farmer and the title character of the non-fiction bookThe Immortal Life of Henrietta Lacks by Rebecca Skloot.

The event is free, and the public is invited to attend.

The Immortal Life of Henrietta Lacks is the 2012-2013 Common Read selection at JCCC. Students from Composition I classes were assigned the book, as were students from the dental hygiene and practical nursing programs.

Sonny Lacks’ appearance is a capstone to months of reading, writing, studying and discussing Henrietta Lacks, the originator of the famous HeLa cells.

HeLa cells are instrumental in medical research, gene mapping, in vitro fertilization and more, yet the woman behind these cells was all but forgotten until Skloot discovered Lacks’ name and history.

Skloot learned that in 1951, Henrietta Lacks unknowingly “donated” cells – both cancerous and cancer-free cells – that had an amazing propensity for growth. The cells were known as the “HeLa” strain, so named after the first two letters of Lacks’ first and last name.

In his appearance, Sonny Lacks will share what it meant to find out – decades after the fact – that his mother’s cells were being used in laboratories around the world, bought and sold by the billions. His visit puts a personal face to big issues such as the dark history of experimentation on African Americans, the birth of bioethics and the legal battles over “informed consent.”

Lorie Paldino, adjunct instructor, English, and chairperson of the Common Read program, said she thought Sonny Lacks’ visit was the perfect way to personalize those issues.

“It’s a great way of getting the family’s perspective,” she said.

The JCCC Common Read Program is in its fourth year. Common read programs have grown in popularity in communities across the nation. Colleges and universities have used such programs to infuse fresh academic and social experiences, promote critical thinking and reflection, and bolster reading beyond the classroom.

Sonny Lack’s appearance is also part of the college’s Scholar-in-Residence program, designed to bring visiting scholars to students, faculty and the public. It is co-sponsored by the English and Journalism division.

 
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Posted by on February 18, 2013 in Education

 

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Crossover events in Prophase I of Meiosis

There are two mechanisms for establishing diversity amongst progeny that are employed in meiosis. The first is independent assortment, which is the random distribution of chromosomes into sex cells, the second is crossover events that randomize DNA between paired homologous chromosomes.

Recall that in Prophase I pairs of chromosomes come together. This will ensure that when the chromosomes are distributed between the two daughter cells produced in Meiosis I each cell gets one of each pair. The pairing forms a structure known as a tetrad (referring to the four chromatids of the paired chromosomes). When the chromosomes are joined in this way it is possible (indeed likely) that there will be breaks that occur and swapping of genetic material from one chromatid to another.

Here’s a good (but dry) animation of this process presented by McGraw Hill:

Thomas Hunt Morgan ran one of the first labs studying crossover events (he was looking at fruit flies). We’ll be looking at his work in my class later in the semester, but here’s a preview that might help visualize what is happening using Morgan’s own sketches. This is the same process as described above, however Morgan only drew one chromatid for each chromosome – this does make the illustration simpler, but keep in mind that there are two chromatids present in the chromosomes of organisms during this stage (Prophase I)

Top – paired chromosomes prior to crossover; Middle – crossover occurring; Bottom – Chromosomes following crossover

 
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Posted by on October 16, 2012 in Education

 

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A simple graphic representation of Cellular Respiration

Here are the several steps of Cellular respiration summarized in a flow chart. I suppose I could have used true flow chart symbols to represent each step and then a decision diamond just after the glycolysis reaction to ask whether oxygen was present (and the aerobic reactions could follow) or absent (leading to fermentation).

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Posted by on October 9, 2012 in Uncategorized

 

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