Flow Cytometry at

Boston University Medical Campus

Introduction to some methods that we offer

Yan Deng (X4-5225), ydeng@bu.edu

Gerald Denis (X4-1371), gdenis@bu.edu

Flow cytometry simultaneously measures and analyzes multiple physical characteristics of single particles, usually cells, as they move in a fluid stream through a beam of light.

Any suspended particle between 0.2 and 50μM issuitable.

Larger particles, solid tissue or clumps of cells must bedisaggregated to be analyzed.

Examples: lymphocytes, protozoa, micron beads, chromosomes

Definitions

The particles in the fluid stream scatter incident light,which reveals internal properties, size and granularity.

The particles also fluoresce; they emit laser light at the interrogation point; this light is picked up by detectors arrayed at a different angle to detectors of scattered light.

Definitions

fluidics

optics

electro

nics

Fluidics

sample

flow cell

laser

waste

~2 x 105 to 1 x 107 cells/ml

sheath fluid

Electronics

event FSC(size)

SSC(granularity)

FL1(green)

FL2(yellow)

FL3(red)

FL4etc

1 500 300 638 840 20 -

2 200 100 245 85 50 -

3 600 800 300 700 30 -

digitize

FluidicsPurpose of the fluidics system:

1. Transport particles in a fluid stream to the laser beam to be interrogated

2. Position the sample core in the center of the laser beam

sheath fluid

samplefluid

‘hydrodynamic focusing’

single file particles

● low flow rate

● narrow sample core

● high resolution

● high flow rate

● wide sample core

● low resolution

Fluidics

Concerns

1. Shear rates for cells: check after you complete a run to ensure that the cells are intact.

2. Larger tips are needed for cell sorting.

Optics

Excitation is accomplished by lasers that emit light at specified wavelengths. The atomic properties of the excitation media define this wavelength for a particular laser.

The laser beam is focused on the sample core; lasers must be fixed in place.

This is the most common and versatile wavelength for excitation of fluorochromes.

Argon blue lasers emit light at 488 nm.

Fluorescein (FITC)

Hoechst 33258 Texas Red

Propidium iodide (PI)

Laser light must overlap with excitation wavelength

Optics

Filters resolve overlapping wavelengths of emitted light

Longpass filter: transmits light of longer than or equal toa specific wavelength

Shortpass filter: transmits light of shorter than or equal toa specific wavelength

Bandpass filter: transmits light only within a narrow rangeof wavelengths

Excitation ofTexas Red is notoptimal with 488 nmlaser

http://probes.invitrogen.com/resources/spectraviewer/

http://www.bdbiosciences.com/spectra/

For more information

Electronics

The electronic system: 1. quantifies the voltage pulse2. converts analog signals to digital values3. performs compensation4. transfers data to the computer for analysis.

Adjusting the voltageof the PMT helps tooptimize captureof desired populations

Electronics – Photomultiplier Tubes (PMTs)

Electronics - Amplifiers

Amplifiers are of two types: linear or logarithmic

Linear amplification is typically used with scatter.

Logarithmic amplification is typically used with fluorescence.

DNA content (Linear detection)

DNA content (Log detection)

Electronics

Threshold is the minimum pulse height above whicha signal will be processed electronically.

Electronics

When a threshold value is defined, only signals with intensities greater than or equal to the value are recorded.

Adjustment of blood immunophenotype to exclude debris

before after

Fluorescence

FluorescenceFluorescence intensity is proportional to the number of binding sitesof the fluorescent compound, or “fluorochrome”.

Remember, when labeling live cells, to keep the experiment at 40C, becausereceptors are internalized and recycled, leaving their fluorochromes behind.

Immunophenotyping

negative stain positive stain

Cluster of Differentiation antibody specificity

CD3 Pan-T lymphocytes CD4 T helper/Inducer CD5 T cells, subset B cells CD11b Monocytes, granulocytes, NK/T cells CD19 B cellsCD20 B cells CD25 Il-2R, Tac IL-2 receptor, Activated T cells,

B cells, NK cells, monocytes CD34 Progenitor cells CD69 Early activation antigen on T, B and NK cells

Immunophenotyping

Two-color fluorescence of lymphocytes

48%

44%

7%

1%

CD19-FITC

CD

3-P

E

Note the experiment-specific definition of the “negative” population

Fluorescence

Multicolor analysis

Spectral overlap

Compensation

To correct for emission spillover of FITC signal (normally detected in the FL1 channel) into the FL2 channel (which detects PE), it is necessary to use filters or electronic compensation or both.

Uncompensated Optimal

Compensation

Before After

Multicolor immunophenotyping

Data presentation formats

Gates

Gates

The uses of gates for cell sorting

Some applications

Multiparameter sorting

Cell cycle kinetics

Some applications

DNA content for cell cycle / apoptosis

Immune system activation