complete basic flow cytometry principles manual

Basic Flow Cytometry

Principles

Presented by: Chrisna Durandt

REVISION STATUS Version 1.0 (Feb 2008)

COURSE DESCRIPTION

During this course the basic principles of flow cytometry will be discussed and

demonstrated. Commonly used flow cytometry terminology will also be discussed and

explained.

TRADEMARKS ALTRA, BECKMAN COULTER Logo, COULTER, COULTER CLENZ, CYTO-COMP,

CYTO-TROL, Cytomics, Elite, EPICS, FlowCentre, Flow-Check, Flow-Count,

Flow-Set, Immuno-Brite, ImmunoPrep, Immuno-Trol, IsoFlow,

Multi-Q-Prep, Q-Prep, T-Q-Prep, XL, XL-MCL and Stem-Trol are Trademarks of Beckman

Coulter, Inc.

COURSE DESIGN Dr. Chrisna Durandt Beckman Coulter South Africa

- i -

BASIC FLOW CYTOMETRY PRINCIPLES

FLOW CYTOMETRY Flow cytometry is the measurement (meter) of single cells (cyto). Flow cytometry

can be used to simultaneously measure and analyze multiple physical characteristics of

single cells/particles.

Let us look at more DEFINITIONS of flow cytometry: A method of measuring the number of cells in a sample, the percentage of live cells in a sample, and

certain characteristics of cells, such as size, shape, and the presence of tumor markers on the cell

surface. The cells are stained with a light-sensitive dye, placed in a fluid, and passed in a stream

before a laser or other type of light. The measurements are based on how the light-sensitive dye reacts

to the light.

www.dana-farber.org/can/dictionary/

Technique for characterizing or separating particles such as beads or cells, usually on the basis of their relative fluorescence.

www.combichemistry.com/glossary_f.html

A process in which cell or particle measurements are made while the cells or particles pass, preferably in single file, through the measuring apparatus in a fluid stream.

www.seagrant.sunysb.edu/BTRI/btriterms.htm

The measurement of cells or cellular properties as they move in a fluid stream past stationary detectors.

health.enotes.com/nursing-encyclopedia/fetal-cell-screen

Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical

characteristics of single cells flowing through an optical and/or electronic detection apparatus.

en.wikipedia.org/wiki/Flow cytometry

FLOW CYTOMETRY A process for measuring the characteristics of

cells or other biological particles as they pass

through a measuring apparatus in a fluid stream.

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FLUORESCENCE MICROSCOPY & FLOW CYTOMETRY A flow cytometer can also be described as a high-throughput, automated fluorescence

microscope.

FLUORESCENCE MICROSCOPE FLOW CYTOMETRY

Error!

Rev: 1.0 (Feb 2008) 2 of 35

SAMPLING AREA Sample presented on slide.

Produce an image of the cell.

Section of whole tissue can be investigated.

Sample is presented in a liquid stream flowing through a sensing area.

Does not produce an image of the cell. Only single cell suspension can be analyzed. To

analyze solid tissues single-cell suspension must

first be prepared.

LIGHT SOURCE Xenon or mercury-vapor lamp Usually a laser(s), but xenon arc or mercur-vapor

lamps are also used on certain instruments

DETECTION Human eye Image might also be projected on computer

screen.

Electronic

OPTICS

DICHROIC mirrors is used on both systems

EMISSION filters are used on both systems


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ANALYSIS Objective interpretation. Depending of the

investigators interpretation

Small number of cells analyzed (hundreds) Slow analysis time 100/sec Low sensitivity: Only strong protein

expression can be detected

Qualitative results: Cells scored as +/-

Subjective interpretation.

103 106 cells analyzed. More accurate High-throughput: 1000 5000 cells/sec High sensitivity: Weak protein expression can be

clearly identified

Quantitative results: Fluorescence intensity of each cell individually scored.


COMPARISON BETWEEN FLUORESCENCE MICROSCOPY &

FLOW CYTOMETRY RESULTS As mentioned before the fluorescense intensity of each cell is individually scored. A flow

cytometry plot is usually divided into 1024 channels in which the fluorescence intensity

levels can be displayed. Therefore it can also be explained that 1024 different levels of

fluorescence intensities can be displayed, resulting in excellent sensitivity.

Rev: 1.0 (Feb 2008) 4 of 35

Increase in fluorescence intensity

FLUORESCENCE 1024 channels

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Each channel represents a specific fluorescence intensity level.

An increase in channel number reflects an increase in fluorescent intensity.


Example: Fibrobrast-like synovial cells (FLS) were transduced with pRET2.ECFP which fluoresces

green. The FLS from patients with rheumatoid arthritis (RA) were transduced with (a) (g)

unconcentrated, (b) (h) 10X concentrated, or (c) (i) 100X concentrated pRET2.EGFP

supernatant. (ac) The percentage of EGFP-positive FLS was determined by flow

cytometry. (gi) Fluorescence microscopy images of cultures following transduction are

shown. These results are representative of data using FLS isolated from 5 RA patients.

Yang et al. Arthritis Res 2002 4:215

Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral

supernatant

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Second peak represents positively stained, green fluorescent cells. Cells show fluorescent intensity between channel 1 and 100. Percentages represent % cells that fluoresces green in relation with all cells counted.

First peak represents unstained cells with no or little fluorescence. Therefore low channel number (0.1 1)


Rev: 1.0 (Feb 2008) 6 of 35

Above-mentioned example also indicates the two main ways flow cytometry results are reported:

% expression Mean Fluorescence Intensity (MFI) expressed as mean channel number.

Absolute number of cells (cells/l) can also be obtained using a flow cytometer and will be discussed later in the course. WHY IS FLOW CYTOMETRY USED? Flow cytometry can be used to measure intra- and/or extracellular characteristics of

individual cells. The following information can be obtained for each individual

cell/particle

Size Complexity / Granularity percentage of a specific cell population in a sample absolute number of specific cells/particles in a sample amount of fluorescence per cell/particle

SAMPLE CRITERIA The power of flow cytometry is its ability to measure multiple parameters of individual

cells within heterogeneous populations. It is not necessary to purify/isolate cells before

flow cytometry analysis.

The sample should be a single-cell suspension. The optimal concentration is 2 5 x 106 cells/ml and Most bench-top flow cytometers require at least 0.5 ml of prepared sample for

analysis.

Most flow cytometers are able to measure cells that are between 0.5 m and 40 m in diameter. Cells, viruses, bacteria, liposomes, etc can be measured using flow

cytometry.


BASIC PRINCIPLES OF FLOW CYTOMETRY Flow cytometry uses the principles of light scattering, light excitation, and emission of

fluorochrome molecules to generate specific multi-parameter data from particles. Cells

are hydro-dynamically focused in a sheath of isotonic liquid before intercepting an

optimally focused light source.

HYDRODYNAMIC FOCUSING In order to make any measurements, you need to have

cells in suspension that flows in single file through the

point of interrogation. This will allow measurements to be

made one cell at a time. The principle of hydrodynamic

focusing is used to achieve this. Sheath fluid flows

through the sensing area known as the flow cell. Within the

flow cell, a slow-moving sample stream is injected into the middle

of the faster moving sheath stream causing the two fluids

(sheath and sample solution), which differ enough in their velocity

and/or density, not to mix. A two-layer stable flow is

formed, with the sheath fluid acting as the wall. By using

the principle of hydrodynamic focusing a much

FOCUSED LASER smaller sample stream can be created,

down to the micrometer magnitude.

Careful control of the velocity of the two streams allow for accurate control of the width

of the sample stream, aligning the cells to flow in single file past the point of

interrogation.

SHEATH FLUID is a balanced electrolyte solution with very specific characteristics,

including:

Non-fluorescent, allowing improved noise-to-signal ratio Low background (filtered to 0.2 m) Bacteriostatic Fungistatic Do not alter the characteristics of the sample, i.e pH, protein expression, etc.

Rev: 1.0 (Feb 2008) 7 of 35


On most bench-top flow cytometers the operator has the option to select between three

(3) sample flow rates:

Low Medium High

The Sample Flow Rate is control by the difference in velocity between the sample and

sheath streams.

SAMPLE PRESSURE = 3.72 p.s.i.

FLOW RATE: 10 l/min

Slow analysis time

Increased sensitivity

LOW


FLOW RATE: 30 l/min MEDIUM


FLOW RATE = 60 l/min

Fast acquisition

Decreased sensitivity

HIGH

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LIGHT SOURCE To be able to make measurements there has to be a light source focused on the

interrogation point. On most flow cytometers the light source is a laser.

Laser is an acronym for "Light Amplification by Stimulated Emission of Radiation

A typical laser emits light in a narrow beam and with a well-defined wavelength,

corresponding to a particular color (i.e., monochromatic).

Depending on the flow cytometer, lasers can be inexpensive, air-cooled units or

expensive, water-cooled units. The air-cooled blue argon laser (excitation: 488 nm) is

the most commonly available laser on single laser flow cytometers. On two laser flow

cytometers the second laser is commonly the red helium-neon (HeNe) laser (excitation:

633 nm) or red diode laser (excitation: 635 nm).

Other lasers that are used on flow cytometers are:

UV helium-cadmium (HeCd) laser (325 nm) UV (HBO) lamp (366 nm) (usually used for DNA work) violet diode laser (405 nm) green helium-neon (HeNe) laser (543nm)


LIGHT SCATTER As the particles/cells pass the laser, light is scattered in all directions. Detectors are placed forward of the intersection point as well as perpendicular (90o angle

with respect to the laser beam). The detector placed in line with the laser path is known as

the Forward Scatter (FSC) detector. Forward scatter is proportional to cell size; the

bigger the cell, the more light is scattered, the higher the detected signal.

Signal to right of plot. High channel number.

FSC

FSC

Signal tLow chann

o left of plot. el number.

Rev: 1.0 (Feb 2008) 10 of 35

Laser Forward Scatter

Detector

Side Scatter Detector

Forward Scatter Detector

Larger particle

Smaller particle

The first detector of a series of detectors placed

perpendicular to the laser path is known as the Side

Scatter (SSC) detector. Side scatter is proportional to

cell complexity/granularity. The more organelles/bits

inside the cytoplasm, the more light scatter, the higher the

detected signal.


Forward and side scatter may be used to identify cell populations of interest.

Rev: 1.0 (Feb 2008) 11 of 35

GRANUCLOCYTES: Increased size, with high complexity

MONOCYTES: Intermediate size, with increased complexity

FOR

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SC

ATT

ER

SIDE SCATTER

LYMPHOCYTES: Small , with little complexity

DEBRIS: Small in size. Little complexity

SAMPLE INFORMATION: Typical, lysed whole blood preparation.

Each dot represents one cellular event.

Although it is possible to analyze platelets

using a flow cytometer, platelets are to

small to be detected using the instrument

The FSC vs SSC plot allows for easy differentiation of small lymphocytes from the larger

monocytes and differentiation of the monocytes from the similarly sized but significantly

more complex neutrophils.


FLUORESCENCE

Other properties of the cell, such as surface molecules or intracellular constituents, can

also be accurately quantitated if the cellular marker of interest can be labeled with a

fluorescent dye; for example, an antibody conjugated to a fluorescent dye may be used to

bind to specific surface or intracellular receptors. Other dyes have been developed which

bind to particular cellular structures (e.g. DNA, mitochondria) or are sensitive to the local

cellular chemistry (e.g. Ca++ concentration, pH, membrane potential, etc.).

Rev: 1.0 (Feb 2008) 12 of 35

CD4+T cell

CD 3

CD 45

CD 4

Depending on the flow cytometer, 3-9 different fluorescent colors may be detected

simultaneously.

Laser

The fluorescent detectors are also placed perpendicular to the laser, after the side scatter

detector. Each detector detects a specific fluorescent wavelength.

Fluorescence detectors(PMT3, PMT4 etc.)


The molecules of flourochromes

(fluorescent dyes) are capable to be

excited, via absorption of light

energy, to a higher energy state, also

called an excited state. The process

is known as excitation (n). The energy of the excited state is

unstable and when stimulation of t

fluorescent compound is stopped (by

removing the exciting light source), it quickly adopts a lower-energy excited state, whic

is semi-stable(o) . Next, the molecules rearrange from the semi-stable excited state bato the ground state, and the excess energy is released and emitted as light. This process is

called fluorescence (p).

he

h

ck

Fluorescence is always of a lower energy, and hence longer wavelength, than the exciting

light, and this separation in wavelength is known as the Stokes shift. The Stokes shift

enables the exciting and emitted light to be separated by optical filters and therefore the

amount of fluorescence can be quantified.

Rev: 1.0 (Feb 2008) 13 of 35


Rev: 1.0 (Feb 2008) 14 of 35

Knowledge of the excitation wavelength is necessary for the selection of the appropriate

parameters.

Thus if you want to use a certain fluorochrome (fluorescent dye), you need to make sure

it is compatible with the laser in use.

Each fluorochrome (fluorescent dye) has a very specific excitation and emission

wavelength. The excitation wavelength is important for laser selection, while the

emission wavelength is important for filter selection.

The magnitude of the Stokes shift varies between fluorescent molecules, and it is

therefore possible to separate the fluorescence emitted by different molecules excited by

the same light source.

FITC: Fluorescein Isothiocyanate

PE: Phycoerythrin

ECD: Phycoerythrin-Texas Red

PARAMETERS Signals (information) you want to acquire.

FS (forward scatter) SS (side scatter) FL1 (fluorescence 1) usually 525 nm FL2 (fluorescence 2) usually 575 nm FL3 (fluorescence 3) usually 625 nm FL4 (fluorescence 4) usually 675 nm etc


NAME EXCITATION

(nm)

APPROPRIATE

LIGHT SOURCE

EMISSION

(nm)

SUITABLE

BAND PASS

FILTER (nm)

Alexa Fluor* 488 Alexa Fluor* 488 495 Blue Diode/Argon Laser 520 525

Alexa Fluor* 647 Alexa Fluor* 647 650 Red Diode/HeNe Laser 668 675

Alexa Fluor* 700 Alexa Fluor* 700 696 Red Diode/HeNe Laser 719 720

AMCA Amino Methylcoumarin Acetic

Acid

354 Hg Arc Lamp or UV

lamp

448 450

APC Allophycocyanin 650 Red Diode/HeNe Laser 660 675

APC-Alexa Fluor*700 APC-Alexa Fluor*700 650 Red Diode/HeNe Laser 719 720

APC-Alexa Fluor*750 APC-Alexa Fluor*750 650 Red Diode/HeNe Laser 780 780

CY*5 Cyanin 5 580 Red Diode/HeNe Laser 670 675

CY*5.5 Cyanin 5.5 630 Red Diode/HeNe Laser 694 700

DAPI 4,6-Diamidino-2-phenylindole 370 Hg Arc Lamp or UV

lamp

455 450

ECD Phycoerythrin-Texas Red* 480 Blue Diode/Argon Laser 620 620

FITC Fluorescein Isothiocyanate 495 Blue Diode/Argon Laser 520 525

FLMA Fluorescein 5-Maleimide 495 Blue Diode/Argon Laser 520 525

PC5 Phycoerythrin-Cyanin 5 480 Blue Diode/Argon Laser 670 675

PC5.5 Phycoerythrin-Cyanin 5.5 480 Blue Diode/Argon Laser 694 700

COMMONLY USED FLUORESCENT DYES

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Rev: 1.0 (Feb 2008) 16 of 35

PC7 Phycoerythrin-Cyanin 7 480 Blue Diode/Argon Laser 767 770

Pacific Blue Pacific Blue 405 Violet/405 nm Diode

Laser

455 450

PE Phycoerythrin 480 Blue Diode/Argon Laser 575 575

PE/CY5 Phycoerythrin-cyanin 5 480 Blue Diode/Argon Laser 670 675

PE/CY5.5 Phycoerythrin-cyanin 5 480 Blue Diode/Argon Laser 694 700

PE/CY7 Phycoerythrin-cyanin 7 480 Blue Diode/Argon Laser 767 770

PI Propidium Iodide 540 Blue Diode/Argon Laser 620 620

RD1 Phycoerythrin 480 Blue Diode/Argon Laser 575 575

Rho 110 Rhodamine 110 496 Blue Diode/Argon Laser 525 525

SPRD SpectralRed* (Phycoerythrin-

Cyanin 5)

480 Blue Diode/Argon Laser 670 675

TRITC Tetramethyl Rhodamine

Isothiocyanate

555 Hg Arc Lamp or Green

Laser

580 575

TXRD Texas-Red-x 575 Hg Arc Lamp or Yellow

Laser

620 620

7-AAD 7-Aminoactinomycin D 550 Blue Diode/Argon Laser 660 675

COMMONLY USED FLUORESCENT DYES


PHOTOMULTIPLIER TUBE The different wavelenghts (fluorescent colors) are separated by optical filters and directed

to sensors called photomultiplier tubes (PMTs). PMTs are light-sensitive sensors that

convert light energy (photons) into electrical current and generate voltage pulse signals,

by converting photons into electrons through the photoelectric effect. The electrons are

directed towards the electron multiplier, where electrons are multiplied by the process of

secondary emission. The voltage pulses generated rise and fall according to the amount of

light entering the PMTs.

Rev: 1.0 (Feb 2008) 17 of 35

micro.magnet.fsu.edu

Filter configuration on EPICS XL-MCL


OPTICAL FILTERS OPTICAL FILTERS

Different types of optical filters are used, each with a very specific function. Different types of optical filters are used, each with a very specific function.

Long pass (LP) filters transmit wavelengths above a cut-on wavelength. For example, a

520 LP filter will allow all light with wavelengths larger than 520 nm trough. All

wavelengths shorter than 520 nm will be blocked out and/or directed to another filter.

Long pass (LP) filters transmit wavelengths above a cut-on wavelength. For example, a

520 LP filter will allow all light with wavelengths larger than 520 nm trough. All

wavelengths shorter than 520 nm will be blocked out and/or directed to another filter.

LLiigghhtt SSoouurrccee TTrraannssmmiitttteedd LLiigghhtt>>552200 nnmm LLiigghhtt

552200 nnmm LLPP FFiilltteerr

Short pass (SP) filters transmit wavelengths below a cut-off wavelength. For example, a

575 nm SP filter will allow light with wavelengths shorter than 575 nm through. All

wavelengths longer than 575 nm will be blocked out and/or directed to another filter.

Short pass (SP) filters transmit wavelengths below a cut-off wavelength. For example, a

575 nm SP filter will allow light with wavelengths shorter than 575 nm through. All

wavelengths longer than 575 nm will be blocked out and/or directed to another filter.

LLiigghhtt SSoouurrccee TTrraannssmmiitttteedd LLiigghhtt


Band pass (BP) filters are used to narrow down the amount of light that is transmitted,

allowing only light within a specific wavelength range to pass through the filter. These

filters are usually used to separate the different fluorescence colors according to their

wavelength properties. For example a 525 nm BP filter will only transmit light between

515 nm 535 nm.

TTrraannssmmiitttteedd LLiigghhttLLiigghhtt SSoouurrccee 662200 -- 664400 nnmm

663300 nnmm BBPP FFiilltteerr

Laser light is very bright and need to be blocked out, to enable emitted fluorescence light

to be detected optimally. A blocking (BK) filter is use for this purpose. The BK filter

completely (99.9%) blocks out light of specified wavelengths, while all other

wavelengths are able to be transmitted. For example, a 457 nm 502 nm BK filter blocks

out wavelengths between 457 nm and 502 nm.

Rev: 1.0 (Feb 2008) 19 of 35


HIGH VOLTAGE & GAIN The voltage pulses generated by the flow cytometer are small and need to be amplified.

The operator is able to manually amplify the pulses by adjusting the

high voltage and/or gain settings

HIGH VOLTAGE is adjusted to change the sensitivity of the light sensor and is applied

directly to the PMT. This adjustment is used to make small, precise adjustments to the

signal. Increasing the voltage of a specific signal will cause a larger electronic pulse to be

generated, resulting in an increase in channel number.

MFI = 500

MFI = 0.5

FL3 MFI: Mean Fluorescence Intensity

FL3 voltage

increase i.e from

350 to 450

MFI = 600

MFI = 0.8 Cou

nt

FL3

Cou

nt

GAIN is the amount of amplification applied to a signal. The signal amplification is done

electronically after the pulses left the PMT. For example, if you increase the gain from

1.0 to 2.0, it will increase the signal by double. Pulses can be amplified 2-, 5-, 10-, 20-,

50- and 75-fold. A single adjustment therefore has a large influence on the signal size and

therefore fluorescent intensity.

Rev: 1.0 (Feb 2008)

MFI = 500

MFI = 0.5

FL3 MFI: Mean Fluorescence Intensity

Cou

nt Adjust gain from

1.0 to 2.0

Signal are doubled

FL3

MFI = 5.0

MFI = 1000

Cou

nt

20 of 35


In flow cytometry, voltages and gains are usually adjusted so that the negative population

within a sample or the isotypic (negative) control falls within the first decade of the plot.

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CD3-FITC

It is very important to note that voltage/gain adjustments have a direct influence on color

compensation settings. Color compensation will covered later in the course. It is

advisable to leave the voltage/gain settings unchanged, while the color compensation is

adjusted.

Rev: 1.0 (Feb 2008) 21 of 35


REGIONS & GATES Regions and Gates are two of the most common used terminologies in flow cytometry.

REGIONS refer to areas of interest on a plot. A region indicates an area of interest from

which statistics will be generated.

For example:

C is a region indicating the CD4 positive cells. Statistics

for the CD4 positive cells will be generated from a to

b.

Rev: 1.0 (Feb 2008) 22 of 35

No statistics will be generated for the negative, first peak as

there is no reference points (generated by creating a region)

from where the statistics should be generated.

C

ba

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CD4 FITC: FL1

Different types of regions may be created.

The following types of regions may be created on two parameter histograms:

Amorphous (polygonal)

Rectangular


Quadrant Regions

Only LINEAR regions can be created in single parameter histograms.

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CD3-FITC

Rev: 1.0 (Feb 2008) 23 of 35


Example:

A. A1, A2, A3, A4, B and C are all regions, indicating specific areas

Total CD4+ Lymphocytes

of interest for which statistics will be generated.

A: Lymphocytes B:

A1: CD4+/ CD3- Lymphocytes C: Total CD3+ Lymphocytes

A2: CD4+/ CD3+ Lymphocytes

A3: CD4-/ CD3- Lymphocytes

A4: CD4-/ CD3+ Lymphocytes

Rev: 1.0 (Feb 2008) 24 of 35


A GATE refers to criteria that must be met before an event is included in a histogram.

ion or

ED

A Gate is applied to a specific plot. It is explained in the following diagram.

A region can become a gate in another plot, therefore are the criteria of inclus

exclusion in other plots

UNGAT

A

SIDE SCATTER (SS)

ALL events that passed the laser are

A

Lymphocytes are the area of interest. Therefore region A

where created around the lymphocytes.

CD4 FITC: FL1

Refers to criteria of what will be further

n

investigated. By assigning A to the histogram, it applies that only what is contained iregion A will be further investigated . Rest of events will be excluded.

This plot is therefore gated on A

Only lymphocytes is included.

oth CD4B - and CD4+ events arepresent as they are all lymphocytes

B

B is a region that included all + t. CD4 T cells the area of interes

included in the plot.

FOR

WA

RD

SC

ATT

ER (F

S)

LOG

SID

E SC

ATT

ER

Rev: 1.0 (Feb 2008) 25 of 35


R

020406080

100

1.00

Cou

nt

Volts Channels

CHANNEL COUNT509 0510 15511 40512 100513 45514 10515 0

ESULTS & STATISTICS meter (histogram) or two-parameters plots. The

he converted pulse for each channel is counted.

tatistics are generated within area indicated.

Results may be presented in single-para

first decade (channel 0 1 (100) usually represents the negative population.

CD3-FITC

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SINGLE-PARAMETER PLOT

A single parameter is used on the x-axis.

CD3 positive

Negative for CD3 The y-axis reflects the number of cells

counted in each channel.

T

S

Rev: 1.0 (Feb 2008) 26 of 35


COUNT: Represents all events (cells) counted within a specific area. If we use the

illustration above it can be explained as all the events counted from

channel 509 515.

MODE or PEAK COUNT: Is the channel with the highest number of events. In the

illustration above, it is channel 512.

MEAN: Refers to the Mean Fluorescence Intensity, therefore the average of all the

fluorescent intensity levels within a specific area.

MEDIAN: Refers to the fluorescent intensity level (channel) which allows 50% of the

events at either side.

50% 50%

Mean

50% 50%

Uneven distribution of

events, therefore the

mean and median

differs.

MedianBecause the distribution is

symmetric the mean and median,

will be similar.

Rev: 1.0 (Feb 2008) 27 of 35


Results may also be presented as a two-parameter plot (dot-plot).

TWO-PARAMETER PLOT

A parameter is plotted on the x- and y-

axis respectively.

Statistics are generated for both the x-

and y-axis.

A1: CD4+/ CD3- Lymphocytes

A2: CD4+/ CD3+ Lymphocytes

A3: CD4-/ CD3- Lymphocytes

A4: CD4-/ CD3+ Lymphocytes

Rev: 1.0 (Feb 2008) 28 of 35


DISCRIMINATOR A channel setting for a parameter that lets you ignore events below the setting. This lets

you eliminate signals caused by debris.

Discriminator

Events below discriminator ignored

Side Scatter

Forw

ard

Scat

ter

Please note that a wrong discriminator, may cause partial or total lost of cells of interest.

Part of lymphocyte population lost

Discriminator

Rev: 1.0 (Feb 2008) 29 of 35


COLOR COMPENSATION

The emission curves of the various fluorescent dyes overlap. This is referred to as

Spectral Overlap. The main aim of the process of color compensation is to separate out

the respective emission curves, therefore decreasing the overlap.

480 500 520 540 560 580 600 620 640 660 680 700 720 740

Rev: 1.0 (Feb 2008) 30 of 35


The use of Band Pass filters placed in front of each of the fluorescent detectors assists in

the segregation of the different signals, but is unable to prevent all of the spectral overlap.

480 500 520 540 560 580 600 620 640 660 680 700 720 740

525 BP

575 BP

625BP

675BP

Color compensation must be performed to ensure signals are indicating their true source

and are not as a result of an overlapping fluorescent signal.

Rev: 1.0 (Feb 2008) 31 of 35


The overlapping signal (interference signal) may cause

Negative populations to be positive

FALSE POSITIVE POPULATION due to incorrect color compensation

Positive populations to present brighter fluorescence intensity than that is true

FALSE INCREASE IN FL2 INTENSITY due to incorrect color compensation

Rev: 1.0 (Feb 2008) 32 of 35


FL1

FL2

FL1

FL2

UNCOMPENSATED COMPENSATED

Colour Compensation is an electronic subtraction of the signals originating from the

fluorescence photo multiplier tubes (PMTs) which enables the correction of the

overlapping fluorescent signals.

There are two options: FL1 FL2 FL2 FL1

Rev: 1.0 (Feb 2008) 33 of 35


Rev: 1.0 (Feb 2008) 34 of 35

L2 is

hat causes FL2 to be more p e?

.

and should be subtracted.

F more positive than it should be. W ositiv It only can be FL1 in a FL1 vs FL2 plot FL1 is therefore the interference signal Therefore it is FL2 FL1

FL1

FL2

Lets compensate the GREEN population first. Is it ?

FL1 FL1 FL2 FL2


The correct adjustment of the colour compensation may be assisted by use of the

atistics, in this example, the Y-mean in quadrants B3 and B4 as shown above. When the

Y-mean is equal in quadrants B3 and B4, then this indicates the correct compensation

ust also be visually

hecked. You can clearly see when a population is under or overcompensated

st

value has been set.

Be careful to depend too much on the numbers. Color compensation m

c

Rev: 1.0 (Feb 2008) 35 of 35

complete basic flow cytometry principles manual

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