j. philip mccoy, jr., ph.d., h.c.l.d. - cytometry · practical flow cytometry, 4th edition howard...

J. Philip McCoy, Jr., PhD

J. Philip McCoy, Jr., Ph.D., H.C.L.D.Email: [email protected]


1 ‐ Introduction 2 ‐ How It All Works

A very brief description of how a flow cytometer works. Please read it – it will help you design your experiments.

3 ‐ Understanding the Lingo A few definitions and explanations

4 ‐ Non‐tradition flow cytometry assays 5 ‐ Tips in Planning 6 ‐ Publishing Your Data – Do’s and Don’ts


The measurement (‐metry) of cells (cyto‐) as they move in a stream (flow) past a set of stationary detectors.

May be performed on a vast array of particles in suspension – e.g. cell, beads, bacteria, organelles.

Generally requires single particle (cell) suspensions (no clumps or aggregates).

ChapterChapter 11


In other words, flow cytometry involves analyzing cells (or particles) which have been placed in suspension. These cells, which have generally been tagged with a fluorescent marker of some sort, are then forced into a narrow stream. The stream is intersected by light from one or more lasers, and the fluorescence from the cells is collected by a set of detectors.


Flow cytometry may not be the appropriate method for all your needs. The main limitations of flow cytometry are the lack of intrinsic morphologic data, and the inability to analyze cell aggregates or tissue specimens. In situations where this is important, consider using alternative methodologies.


Immunohistochemistry if morphology is needed

Confocal microscopy for adherent cells and tissues

Imaging flow cytometry Combines elements of traditional flow cytometry with imaging

PCR Genomic information may prove useful in many circumstances


Flow cytometry is a unique methodology that should be considered for use when: Rare event analysis in which large numbers of individual cells need to be analyzed

A correlated analysis of as many as 20 parameters of individual cells is desired.

High throughput is needed Correlation of cell cycle with immunophenotype


Luminex multibead array For soluble analytes such as serum cytokines and chemokines Can measure up to 100 different analytes simultaneously on as little as 50ul of sample.


Amnis Imagestream Provides images of all cells in addition to tradition flow cytometry data

Technology involved is slightly different than traditional flow cytometry

Data acquisition is slower, and no sorting can be performed.

0 1 2 3 4

0

30

60

.Freq

uenc

y

Bright Detail Similarity GFP-Mitotracker Deep Red

1174/1417 (83%) Co-localized

3/1367 (0.2%) Not Co-localized

Co-localized


A Brief Description of How a Flow Cytometer Works

ChapterChapter 22

To the casual user, the flow cytometer appears to be a complex, somewhat random array of wires, tubes, and gadgets.

Remembering our definition of flow cytometry, it is useful to think of a flow cytometer in terms of its “organ systems”


Fluidics (provide the flow) Lasers (provide the light source) Optics (collect the light signals) Electronics (convert the light signal into digital information)

Computer (for control of the instrument and interpretation and visualization of data)

Necessary for pushing the cells past the detectors.

The stream needs to be precisely aligned with laser and optical path.

Air bubbles or clumps of cells will alter the stream and effect data.

Can be made into droplets for sorting.

SampleSheath stream

Excitation laser

Light Scatter

Fluorescence signal

A coaxial stream is used to push the sample past the laser.

The outer stream is the sheath fluid (generally isotonic PBS).

The inner stream is the sample fluid.

A coaxial stream allows the cells to flow with less turbulence, and directs the cells to the center of the stream (hydrodynamic focusing).

Sheathfluid

Samplefluid

Flow Cell

Sample (Cells)

Emission wavelength of laser must match excitation wavelength of fluorochrome.

Argon , Krypton, HeNe are most common lasers.

Some lasers can be tuned to different wavelengths.


Light fluorescing from the cells must be collected. Fluorescent light emitted from the cells must be distinct and separated from the laser light.

In multi‐color experiments, each ‘color’ of light must be collected separately.

Mirrors, filters, and beam splitters are used to direct and separate the different wavelengths of light.

The next figure illustrates how this occurs.


Optics of a Clinical Flow CytometerFL1

Red Diode LaserRed Diode Laser~635 nm~635 nm

SSC

FL3

FL4

670nm LP

661nm/16BP

585nm/42BP

488nm/10BP

90/10 Beam splitter

DM 560nm SP

Fluorescencecollection lens

DM 640LP

Half mirror

488/10488 nm488 nm

Blue LaserBlue Laser FSC diode

Focusinglens

FL2

530nm/30BP

FlowCell

FlowCell

Modified with permission from BD Biosciences


In addition to fluorescence, the cytometer can also collect light scatter measurements.

These measurement are how the laser light is scattered by the individual cells.

Generally, two types of light scatter are measured: Forward light scatter This is along the axis of laser illumination, and is roughly proportional to cell size, assuming the cell is spherical.

Side (orthogonal) light scatter This is general measured at 90o to the laser path (although it can be at other angles), and provides information concerning the complexity or granularity of cells.


Fluorescence signals must be converted to analog pulses (by photomultiplier tubes –PMTs), and digitized (by analog to digital converters [ADCs]) for computer analysis.

May be used for both control of the cytometer and analysis of data.

Requires digitized data for analysis – termed “listmode files.”

Analysis of data is possible on computers not connected to cytometer.


In order to ensure that all of these systems are operating properly and in concert with each other, rigorous quality control and maintenance procedures are performed on the cytometer.

Many of these procedures are performed daily, others at various intervals


Much of the quality control involves running beads of various types. These can be used to align the stream/laser/optics with each other or to check that fixed alignment remains accurate.

These procedures can take time. Be patient. If the instrument is not set up correctly, your data will be flawed.

Practical Flow Cytometry, 4th EditionHoward M. ShapiroISBN: 0471411256736 PagesDec 2003$167.00 (or free from Beckman Coulter at

http://www.coulterflow.com/bciflow/practical.php) This is a VERY technical, but very well-written

book. Considered to be the “bible” of flow cytometry


Having this basic understanding of how a flow cytometer works should assist you in preparing and running your samples. Realize that a poorly prepared specimen will give poor results, even with a well maintained cytometer. Similarly, a poorly maintained cytometer will yield poor results for even the best prepared specimen.


A Few Definitions and Explanations

ChapterChapter 33


As with most areas of technology, flow cytometry has its unique vocabulary. New users of flow cytometry may be confused when asked about gating or compensation or PMTs. Let’s demystify some of this.


The typical method of viewing flow cytometric data is via univariate or bivariate displays, or histograms. The former displays the fluorescence intensity of the cells along the X‐axis and the number of cells on the Y‐axis. The latter, bivariate plots, display the fluorescence intensity of two stains along the X‐ and Y‐ axes. The number of cells is indicated either by dot density or by using contours.


Bivariate

Univariate of X axis parameter


When analyzing two parameters using a dot plot, lines may be drawn which divide the plot into quadrants. The software will provide statistics on each quadrant, such a the percentage of positive cells, the mean fluorescent intensity of the cells, the coefficient of variation of the staining, and so forth.

FL1 – FITC MAb

FL2

PI

FL1 – FITC MAb

Wrong Right

Gating is a way to tell the cytometer to only examine a portion of the data being collected. This permits heterogeneous cell populations to be put on the cytometer when analysis is only desired on one of the subpopulations. The next slide illustrates this with peripheral blood. Gates are set, or drawn, either during acquisition of data or during analysis (preferred). Multiple gates may be used simultaneously using Boolean logic. Subsequent histograms may be drawn of only the gated cells.


Ungated bloodLight scatter

CorrespondingFluorescence

Gated lymphocytesLight scatter

CorrespondingFluorescence


Multi‐color flow cytometric experiments are complicated by the fact that fluorochromes often have some overlap in their emission spectra. A small proportion of FITC, which we see as green, will be detected in the orange detector. Similarly, phycoerythrin (PE), which we see as orange, will yield a small signal in the green detector. This is termed “bleed over” of fluorescence or fluorescence “crosstalk”.


FITC PE


How data appear if under-, over-, or properly compensated.

Under compensatedmay falsely appear to be dual positive

Over compensatedmay miss dual positive cells since cells are forced too far to the axis

Proper compensationminimizeson-axis events


Cluster Designations are system of standardized nomenclature for monoclonal antibodies. These have been established by a series of international workshops.

Several hundred “CDs” have been agreed upon.

Several monoclonal Abs may have the same CD, but may recognize different epitopes on the same antigen (e.g. various clones for markers such as CD34 or CD14 differ widely in the epitopes recognized). Thus, not all CD34 Mabs will stain cells identically, nor will all CD14 clones stain cells identically. In general it must not be assumed that all clones with a common CD will stain cells identically.


Whenever possible refer to monoclonal antibodies by the CD, not by the individual clone. However, when publishing, including the clone name is recommended.


Today, most flow cytometry data are collected as listmode files in .fcs format. This means that information is stored for all parameters measured on each individual cell in a correlated manner. In other words, it is ‘raw’ data and you can use this to ‘replay’ experiments, change gates, and sometimes even change compensation.


.FCS files are flow cytometry standard files, and represent a somewhat standardized format for the listmode data (although there is some variation between instrument manufacturers in these files).

.FCS files can be reanalyzed including regating and, in some instances, resetting compensation values.


All flow cytometry requires that cells be forced through a small orifice referred to as a flow cell, or nozzle tip. These vary in size, but for most applications are about 100‐200

A cell 10 microns in diameter flows well through a flow tip with a 70 micron opening.

Ten or twelve of these cells in a clump will not.


Beyond Immunophenotyping

ChapterChapter 44


Flow cytometry has traditionally been used for the study of cell surface markers and for DNA/cell cycle analysis.

Flow cytometry can be used for: Intracellular antigens Soluble cytokines or other ligands PCR and FISH Green fluorescent protein transfection Quantitation of antigen expression


T cell receptor alpha and beta variable chains. Cytokine receptors. Fetal hemoglobin in fetal red blood cells and f cells. FRET (energy transfer) analysis.


Fluorescent particles

Phagocytosed by a nonfluorescent cell

Will make that cell fluorescent

Some drugs (eg adriamycin) will fluoresce at specific wavelengths.

Cells which uptake these drugs become fluorescent.

Drug uptake or efflux can be monitored (multidrug resistance [MDR] can be assessed this way).

No MDR,cells with adriamycin fluoresce

MDR pumps outadriamycin

Cells become less fluorescent


SAC permits analysis of cell‐free proteins or ligands (such as cytokines) using beads and ‘capture’antibodies.

As many as 100 spectrally distinct beads analyzed concomitantly, although 5‐10 distinct sets of beads is more common.

Commercial kits available.


Multiplex Bead Array AssaysMultiplex Bead Array Assays


Multiplex Bead Array AssaysMultiplex Bead Array Assays

Beads with different fluorochrome ratios would have distinct “spectral addresses in 2 parameter histograms

These fluorescent parameters are used for gating beads of interest


Flow Cytometry can be used with molecular biology techniques. FISH In situ PCR (eg for detection of HIV) Telomere length measurement GFP or YFP detection


Or How to Keep Your Data Flowing

ChapterChapter 55


A key to obtaining good data in flow cytometry experiments is the following equation:

Garbage in = Garbage Out

In other words, a poor specimen or sloppy staining or preparation methods will always yield poor data.


Label your tubes!!! Check to make sure that the tube size or microtiter plate that you are using to prepare your samples is compatible with the cytometer

CONTROLS ,, both positive and negative, will make instrument setup and data interpretation much easier. Compensation controls are needed to accurately set compensation.


Assess the viability of your specimen. Nonviable cells can nonspecifically bind antibody and yield confusing results. Trypan blue may be used to manually determine viability, but it is even better to add a real‐time viability stain to your samples for analysis on the cytometer. If a high proportion of dead cells are found in the specimen, it may be possible to remove them by gradient separation or by simply gating them out if you have performed real time viability staining.


An example of a real time viability stain would be propidium iodide (or 7AAD) that stains only dead cells and permits them to be gated out of further analysis.

If you use propidium iodide remember that it has a wide emission spectrum and may interfere with other fluorochromes that you plan on using.

J. Philip McCoy, Jr., PhDFL1 – FITC MAb

FL2

PE

A population with fluorescence like this (dual + at a 45o angle)is likely due to dead cells


The best solution to the problem of cell clumps is to filter samples before they are placed into a cytometer. 50 micron pore nylon mesh tubing is the most often used.

Alternatively, large clumps can be removed by allowing them to settle to the bottom of a test tube and removing the cells remaining in suspension.

Finally, EDTA may reduce the formation of clumps, although it may be less effective in dissolving them.

Flow cytometry is based on the premise of single cell analysis.

A cytometer may recognized two cells stuck together as one large cell.

This would look like one double positive cell.


FL1 – FITC MAb

FL2

PI

FL1 – FITC MAb

FL2

PI

This population could be a clump of red and green cells


Not all fluorochromes can be run on all flow cytometers. This is partially dependent on which laser(s) light source is on the cytometer.

Not all fluorochromes can be run with each other due to spectral overlap.

Some fluorochromes are brighter (PE) than others (FITC) (due to better quantum yield). The brighter fluorochromes are better to use if you are expecting dim staining or if you have observed dim staining with other fluorochromes.


Fluorochromes which are generally usable: Green – FITC or GFP (525nm emission) Orange – PE or PI (585nm emission) Red – PerCP or PE‐Cy5 tandem conjugate (680nm emission) Red (with red laser) – APC or Cy5 (660nm emission)

Many other fluorochromes can be used! Check with the flow lab before staining if you are uncertain.


This is one of the most frequently asked questions. There is no one universal answer for this question. It is usually best to estimate the number of cells you need for an assay by asking the following: How rare is the population of interest? Do you have instrument settings or does some of your sample need to be used to set these?

What is your cell loss during processing and staining?


Do’s and Don’ts

ChapterChapter 66


Checklist: Type of specimen (ATCC # , etc if cell line). Density gradient separation or RBC lysis? Dyes and fluorochromes used.Monoclonal antibodies Conjugations Clone name and CD if applicable

Washes and centrifugations. Buffer and fixatives used. Length and temperature of incubations.


Checklist:

Type and model of cytometer. Laser (and wavelength) used for excitation. Optics for collection of fluorescence. Log or linear amplification? Number of events (cells) collected/analyzed. Data collected gated or ungated?


Checklist: Software (and version) used for acquisition.Software (and version) used for analysis.Any smoothing or altering of histograms or plots.


In general the compensation values do not have to be published.

Notation should be made if figures are presented as compensated data.

State if compensation was set before the data were collected or during analysis.

Axes should not be labeled “Fl1” or “Fl2” unless more information is supplied.

It is best to label the axes with the fluorochrome and antibody as shown on the right.

Always show tick marks to indicate the scale.

NEVER rely solely on isotype controls to set markers or assign quadrants in order to determine the percentage of positive cells.

IsotypeControl

Experimental

Would you reset the quadrants?

Populations of cells should NOT be split unless absolutely necessary.

Quadrants can be set based on the clustering of cell populations. This better describes the biology.

Wrong

Right


A good understanding of the basics of flow cytometry will foster good practices in daily work.

This must be accompanies by a good understanding of the biology of the cells you are analyzing.

For immunophenotyping, understanding antigen‐antibody interactions is a must!

When in doubt, ask for help…remember flow cytometry can generate data anyway you run the instrument – but GOOD data only comes from GOOD understanding and practices.


Practical Flow Cytometry, 4th Edition, Howard M. Shapiro (http://www.coulterflow.com/bciflow/practical.php)

Compensation Website: (http://www.drmr.com/compensation/)

Invitrogen flow cytometry tutorial (http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html)

Flow Cytometry - A Basic Introduction, Michael G Ormerod (http://flowbook.denovosoftware.com/)

j. philip mccoy, jr., ph.d., h.c.l.d. - cytometry · practical flow cytometry, 4th edition howard...

Documents