Flow Cytometry - cdn1.sinobiological.com

Flow Cytometry

- The Essentials and Its Applications in

Cancer Research

Contents

Introduction to Flow Cytometry

Principle of Flow Cytometer

Tips & Tricks for Flow Cytometry

Applications in Cancer Research

Flow Cytometry Troubleshooting

Introduction to Flow Cytometry

1

Definition of FCM

Flow Cytometry (FCM)

A technology for multi-parameter analysis,

rapid quantitative analysis or sorting of rapidly

flowing cells or biological particles with Flow

Cytometer.

Features of FCM

Multi-

parameter

High

Sensitivity

High

ThroughputQuantitative

Sorting

Comparison of flow cytometry with fluorescent microscopy：

Fluorescent microscopy Flow cytometry

View, Still, Simultaneous Single, Flow, In turn

Eyes/CCD PMT

Internal structure information of

cells

Cell population characteristic

distribution

FCM: Single cell analysis, collecting data

for each cell

WB: Population analysis, yielding an average

of multiple cells

FCM vs. WB

Principle of Flow Cytometer

2

Classification of Flow Cytometers

Flow cytometric analysis & Cell sorting

Cell Analyzers Cell Sorters

After analysis, sample finally enters the waste liquid

tank and cannot be recycled.

Components of Flow Cytometer

Fluidics system

· Flow chamber

· Flow drive system

Optics system

· Laser source

· Light collection system

Electronic system

· Signal detection system

· Data processing system

Fluidics System

Hydrodynamic

Focusing

Laser Focus

Sample

Sheath

Fluid

Flow

Chamber

Flow Chamber

It’s the core component of the instrument where the sample is measured with the laser. The

flow chamber is made of quartz glass where a rectangular hole with a diameter of

430μm*180μm is located in the center allowing only single cell to flow through. The detection

zone is at the center of the hole. The good optical characteristics and slow flow rate of the

chamber make cells expose to laser sufficiently. Sufficient light flux of the cells can be

collected. With wide-angle collection lens, high detection sensitivity and measurement

accuracy can be obtained.

Laser Beam

Flow Drive System

Sheath Fluid Technique: Two liquids coaxially flow, and the sample cells flow in the center.

Hydrodynamic Focusing: There is a focus contraction effect when the steady liquid flows

from large to small cross-sectional area. The cell flow diameter is constrained to about 10-

20μm to ensure only single cell to pass through the detection zone.

Focuses the cell suspension, causing cells to pass through a laser beam one cell at a time.

Detection

Components of Flow Cytometer

Optics System

Laser

Laser is a kind of coherent light source that provides single wavelength,

high-intensity and stability illumination. It is an ideal source for rapid

analysis of weak fluorescence of cells.

The laser beam illuminates the cell flow at a 90° angle, and the cells

produce scattered light and fluorescence.

Light Collection System

Optical filters send different wavelengths of fluorescent signals to different

electron detectors.

Long Pass Filter (LP): Allows beams above a certain wavelength to pass;

Short Pass Filter (SP): Allows beams under a certain wavelength to pass;

Band Pass Filter (BP): Allows beams within a certain wavelength range to pass.

Long pass Short pass Band pass

460 500 540

SP 500LP 500 BP500/50

460 500 540 460 500 540

Components of Flow Cytometer

Electronic System

Converts an optical signal into an electrical signal and digitizes it for computer analysis:

Photoelectric detection: Converts an optical signal into an electrical signal.

Preamplifier circuit: Proportionally amplifies the signal and outputs an electrical pulse signal.

Analog to digital converter: Converts an analog electrical pulse signal into a digital signal.

Data processing system: Computer system data acquisition and analysis.

Components of Flow Cytometer

Signal MeasurementScattered Light Signal

Granulocyte

Monocyte

Lymphocyte

Red blood cells, Dead cells and Debris

In experiments, the combination of FSC and SSC parameters is often used to distinguish different cell populations and remove

interference from debris, dead cells and adhesion cells.

Dead cells,

Debris

Adhesion cell

Intrinsic Parameters

Forward-scattered light (FSC): It is the isotropic light signal of an incident

laser that reflects the relative size and surface area of the cells.

Side-scattered light (SSC): It is the scattered light signal at a 90° angle of

the incident laser , reflecting the intracellular complexity.

Fluorescent Signal

Autofluorescence: The cell itself emits a weak fluorescent signal

forming the noise signal under illumination of the laser;

Specific fluorescent signal: The fluorescent signal obtained by

laser illumination after the cells are labeled with fluorescein.

Signal Measurement

Each fluorescent dye produces a specific wavelength of fluorescence

and color, and a wavelength-selective permeability filter separates the

fluorescent signals of different wavelengths into different

photodetectors.

Multiple different traits on one cell can be determined simultaneously

by selecting different monoclonal antibodies and dyes.

Tips & Tricks for Flow Cytometry

3

Experimental Design

• Confirm the markers in the experiment

• Select the appropriate fluorescein-labeled

antibody

Ⅰ. Specificity of Target Protein

Ⅱ. Correct Species

Ⅲ. Prefer Directly Labeled Antibodies

Ⅳ. Applicable for FCM

Experimental data graph

Experimental DesignFluorescein Selection

Intensity of Fluorescein Spectral Overlap

Fluorescein:

Some substances emit light with a wavelength longer than the wavelength of the illuminating light in a specific

wavelength range. This kind of light is called fluorescence, and the substance that generates fluorescence is

called fluorescein.

Experimental Design

Fluorescein Selection

Fluorescence matches the instrument

Laser, number of detection channels and specific

parameters

Ⅰ

• Only one fluorescein can be selected per channel;

• The fluoresceins between the channels can be

arbitrarily matched.

Fluorescein brightness matches the

antigen expression level

Antigen expression level & fluorescein brightness

Ⅱ

• High level expression Weak fluorescein

• Low level expression Bright fluorescein

Consider spectral overlap using

multicolor fluoresceinⅢ

Excitation and emission spectra of fluorescein

More Matters

• Some fluoresceins can be excited by multiple lasers;

• Tandem dyes are easily degraded;

……

Ⅳ

Features of fluorescein

• Minimize the spectral overlap of antigens expressed

on the same cell;

• Multiple laser excitations are used to reduce spectral

overlap.

Sample Preparation

Peripheral

blood

Marrow

Tissue

Cultured

cells

Exfoliated

cells

Principle：

Ensure high quality single cell

suspension, minimizing debris and

dead cells

Fluorescence StainingSet Control Experiments

Single Stain

Control

Ⅱ Ⅲ

Isotype

Control

Ⅳ

FMO

Control

Adjust instrument

compensation

Exclude the non-specific

staining background of specific

subtype antibodies

FMO control is the sample

containing all required

antibodies minus one of

them. Any spread of the

fluorochromes into other

channel can be properly

identified.

Ⅰ

Negative

Control

• Indentify the background

and the level of

autofluorescence of the

cells;

• Autofluorescence

interferes some

fluoresceins such as FITC

and Pacific Blue, reducing

signal resolution and

causing false positives.

FL-1(FITC)

FL-2

(-)

FITC

single

stain

FL-1(-)

FL-2

(PE

)

PE

single

stain

Fluorescence StainingOptimize Experimental Details

Antibody titer

Ⅰ

Mouse splenocytes were stained with anti-CD27 FITC (50110-R012-F) in multiple concentrations

Antibody titer method：

Use the same kind of cells in the experiment; Fix the cell amount and reaction

volume; Add different amounts of antibodies and calculate the positive peak

fluorescence intensity and signal to noise ratio (S/N).

1. Wide range of titer density, 5 gradients are better.

2. The experimental conditions of the titration should be consistent with the

actual experimental conditions, such as the reaction temperature and pH.

3. Weakly expressed/ungrouped antigens are not suitable for titration.

Eliminate Interference of Dead Cells

Autofluorescence of dead cells is high;

Dead cells can easily ingest antibodies, leading

to non-specific binding. The experimental results

will be affected;

Dead cells may release DNA, which is very

viscous and can cause cells to clump and easily

block the tube.

Live

All

Distinguish Dead Cells

1. DNA-binding dyes: DNA dyes such as PI,

DAPI, 7-AAD, and TO-PRO-3 can only enter

cells whose membranes are damaged and bind

to the DNA of those cells.

2. Protein-binding dyes: Bind to the free amine on

the cell surface or inside the membrane. The living

cells are weak positive, and the dead cells are

strong positive.

They can be used in fixed specimens to distinguish

the state of fixed cells in the sample.

Representative products: live/dead dyes

Fluorescence StainingOptimize Experimental Details

Ⅱ

Features of dead cells: Enhanced membrane permeability and loss of

membrane integrity

Block Fc Receptor

Fc receptors are proteins on the cell surface that bind to

immunoglobulins (IgG, IgA, IgM, IgE, and IgD), existing

on the surface of NK cells, mast cells, macrophages and

neutrophils. Fc receptors can bind to Fc fragment of

antibody, leading to false positive results.Before blocking

After blocking

(1) Commercial Fc block reagents:

Recombinant human IgG, mouse CD16/CD32 antibody

Fluorescence StainingOptimize Experimental Details

Ⅲ

Blocking Reagent of Fc Receptors

(2) Human serum or serum of specific species

Detection

How many cells

should be obtained

in my experiment?

How many cells can

make sense?1. If the target cell population is high, 10,000 cells need to be detected；

2. If the target cell population is low (under 5%)：

Obtain Cell Amount

Data reality: Eliminate non-specific signal caused by dead cells and debris;

Complete control experiment;

Adjust the amount of cells obtained: Repeat the experiment several times;

Collect the same number of cells each time; Calculate the CV value. If you want

decrease the CV value, you can try to collect more cells.

Data AnalysisData Display Form

3D ImageTwo-parameter

data display

Single parameter

data display

Distribution histogram

Each single parameter data can be

organized into distribution statistics

and displayed by distribution histogram.

Abscissa: relative intensity of the

fluorescent signal or the scattered light

signal.

Ordinate: relative cell number

DNA ploidy analysis

Dot Plot

Density Plot

Contour Plot

CD

8

Mean Fluorescence

Intensity (MFI)Absolute CountingPercentage

Data AnalysisData Analysis Method

The percentage comparison method

is based on the same number of

parent cell populations.

2, Flow cytometer with absolute

counting function:

Precisely control the sample volume;

Calculate the total number of

specific cells per unit volume by the

number of collected cells, sample

volume, total volume, and absolute

number.

1. TruCount tube: The tube contains a

known number of microspheres;

1. Mean: Arithmetic mean for linear

positive distribution;

2. Geometry Mean: Geometric mean

for lognormal distribution;

3. Median: Median is not affected by

the distribution of data patterns;

4. Mode: Where the highest peak of the

data is located

1. For bimodal cell populations, a percentage analysis is more meaningful;

2. Focus on the absolute number of cells;

3. MFI analysis: Not suitable for bimodal cell populations. Compare MFI among different experiments cautiously.

2. Flow cytometer with absolute

counting function:

Precisely control the sample volume;

Calculate the total number of specific

cells per unit volume by collecting the

number of cells, sample volume, total

volume and absolute number.

Note：

Flow Cytometry Troubleshooting

4

No Positive Signal / Weak Signal

Detect internal antigen.

Whether the cell membrane

is fully permeabilized;

Whether the surface

molecules have been fixed

before fixation.

No positive

signal /

Weak signal

Identify antigen expression

level.

Antigen

Whether the antibody is matched

with the instrument;

Whether the antibody is

correctly stored;

Whether the antibody concentration

is appropriate;

Whether the secondary antibody is

right in indirect staining.

Antibody

Sample preparation

Use bright fluorescein if antigen

expression is low;

Amplify signal.

Antigen

Change antibody;

Store at 2-8℃, protect from light

and avoid freezing ;

Adjust antibody concentration.

Antibody

Replace cell permeabilization

method ； Some molecules are susceptible to

fixation or enzymatic digestion. Avoid

fixation before surface staining.

Sample preparation

Non-specific StrainingSample

Whether the cell viability is

too low;

Whether the cell expresses

Fc receptor.

Antibody

Whether the concentration

used is appropriate;

Whether the secondary

antibody cross-reacts with the cell

in indirect staining process;

Whether tandem dyes is used.

Sample preparation

Whether it is adequately

washed, especially in

intracellular staining.

Non-specific

Straining

Use reactive dyes to eliminate

interference;

Use Fc receptor block reagents.

Sample

Adjust antibody concentration;

Change secondary antibody;

Change fluorescein.

Antibody

Fully Washed

Sample preparation

Applications in Cancer Research

5

Research Hotpot of Cancer

Tumor microenvironment

Tumor stem cell research

Immunotherapy

Apoptosis and tumor

Angiogenesis and tumor

FCM Applications

Surface antigen;

Cell membrane fluidity and permeability;

Membrane potential;

……

Cytoplasmic protein;

Enzyme activity;

Ca2+ ion content;

……

Nucleic acid content;

Nuclear protein;

……

Almost all components or changes in the cell that can be labeled with a fluorescent dye can be detected

by flow cytometry.

Cell nucleus:

Cell membrane:

Cytoplasm:

FCM Applications

Immunophenotypic Analysis

The differentiation process of T cells: TN

gradually differentiates into TSCM and TCM. These

cells can self-proliferate and further differentiate into

TEM and TE cells with shorter survival time.

In CAR-T therapy, the selection of less differentiated

TN, TSCM or TCM as a raw material for genetic

engineering may result in more effective engineered

T cells.

CD

8S

SC

CD122CD95CD45RO

CC

R7

CD

62

L

CD4

CD3 FSC-A

FS

C-H

FSC

SS

C

Changes caused by apoptosis

1 Mitochondrial membrane potential reduction

3 Phosphatidylserine (PS) eversion

2 Mitochondrial pores (mtPTP) open

4 Caspase activation

5 Cell membrane permeability change

6 DNA break

JC-1 detection

Annexin V detection

Cytochrome C detection

Caspase detection

PI/7-AAD detection

Tunel detection

Detection

Apoptosis refers to the programmed cell death that is controlled by genes to maintain homeostasis.

Apoptosis Detection

Apoptosis process:

Receiving apoptotic signals

Interaction among apoptosis regulatory molecules

Proteolytic enzyme activation (Caspase)

Continuous reaction process

Apoptosis Detection

Annexin V/7-AAD

Detection

Control 5µM Camptothecin 4h

Catalog Number Product Name Size

APK10448-F Annexin V-FITC/7-AAD Apoptosis Detection Kit 20T/100T

APK10448-P Annexin V-PE/7-AAD Apoptosis Detection Kit 20T/100T

Apoptosis Detection

Decreased mitochondrial membrane potential is a landmark event in the early stages of apoptosis.

Many dyes such as JC-1, mitotracker red, rhodamine 123 can be used to detect this change.

Taking JC-1 as an example, when the mitochondria are in a normal state, JC-1 aggregates and is detected to emit red light.

When the membrane potential drops, JC-1 exists in a monomeric form, and is detected to emit green light.

Control 50µM CCCP 20min

Mitochondrial Membrane Potential Detection

Calcium Flow DetectionCalcium acts as a second messenger involved in many biological processes, such as cell metabolism, proliferation, differentiation,

apoptosis, secretion, migration, nerve conduction, fertilization and development. The occurrence of these biological processes is

closely related to the intracellular calcium ion concentration.

Fluo-4 AM is a fluorescent dye that can penetrate cell membrane. Its fluorescence is very weak and does not enhance with increasing

calcium ion concentration. Upon entering the cell, Fluo-4 AM can be cleaved by intracellular esterase to form Fluo-4, which is retained

in the cell. Fluo-4 can bind to calcium ions and combine with calcium ions to produce strong fluorescence. It can be excited by 488nm

laser with an emission wavelength of 525-530nm.

Indo-1 and fluo series of fluorescent indicators can be used to detect intracellular calcium ions.

Cell Proliferation

CFSE can easily penetrate the cell membrane, releasing the fluorescent substance after hydrolyzing in the cell. After

covalently binding to the intracellular protein, it no longer has membrane permeability.

During cell division and proliferation, the fluorescence intensity of CFSE decreases with the division of cells, and the

labeled fluorescence can be evenly distributed to the two daughter cells. Therefore, the fluorescence intensity is half

that of the parental cells.

Principle：

Divisions:

3 2 1 0

Divisions:3 2 1 0

CFSE

FCM

Ⅰ

Our

Team

10+ years’ experience of FCM antibody

screening and development.

Scientificity, accuracy and reliability are

ensured in experimental design, sample

preparation, and data analysis.

Ⅱ

Reagent

Storage

1000+ FCM antibodies are made in

house, as well as Annexin V/7-AAD

apoptosis detection kits.

A variety of commonly used reagents

for flow cytometry are stored, greatly

saving your time and cost.

Ⅲ

Cell

Storage

Provides 200+ cell lines,

eliminating the risks in cell

sample delivery.

A B

E D

F C

Cell surface antigen assay

Intracellular antigen

Cytokine assay

Cell activity assay

Apoptosis assay

Cell proliferation assay

Flow Cytometry Service of Sino Biological

12,000+

Antibodies

6,000+

Proteins

500+

ELISA Kits28,000+

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