Lab 3 - Flow Cytometry

Question 1

What is flow cytometry?

Answer 1

A very powerful technique that enables researchers and clinicians to analyze the properties of single cells within a population of cells

Question 2

Why is flow cytometry commonly used in clinical diagnosis?

Answer 2

It distinguishes the presence of specific cell types or cells exhibiting specific markers

Question 3

How is flow cytometry employed as both a diagnostic and prognostic tool for the analysis of lymphomas and leukemias?

Answer 3

Specific antibodies and fluorophores can be used to identify the presence of cell surface markers characteristic of these malignancies

Question 4

What are the major areas of use for flow cytometers?

Answer 4

Determination of ploidy and cell cycle analysis

Question 5

What basic systems do all flow cytometers have in common?

Answer 5

1) Fluidic system
2) Illumination source
3) Optical and electronics system
4) Data storage and computer control system

Question 6

What does a fluidic system do?

Answer 6

Determines how light meets and interacts with cells or particles

Question 7

What typically occurs in the fluidic system?

Answer 7

• Single particle sample suspensions are directed into a diluent (sheath fluid) and forced into a chamber by air pressure
• While the sample travels by laminar flow through the chamber, the pressure of the sheath fluid aligns the cells and/or particles in single file

Question 8

What will the illumination source of the flow cytometer do?

Answer 8

Project a beam of laser light through the continuous liquid stream of sheath fluid buffer to illuminate cells in suspension

Question 9

Why are lasers used as the illumination source?

Answer 9

Because the beams remain compact with little divergence, they have high spectral purity (single wavelength), and they are excellent excitation sources

Question 10

What is done to cells in order to facilitate the measurements of biological properties?

Answer 10

Cells are often conjugated to fluorescent probes that bind to specific cellular constituents

Question 11

What are the monoclonal antibodies conjugated to?

Answer 11

• Fluorochromes,
• Fluorescent stains (e.g., propidium iodide)
• Transgenes that are fused to a reporter (e.g., green fluorescent protein)

Question 12

When are fluorescent probes excited?

Answer 12

As cells individually pass through the laser path at a rate as high as 1000 cells per second

Question 13

What do excited fluorescent probes result in?

Answer 13

The emission of light at detectable wavelengths

Question 14

What amount of fluorescence is emitted as a cell passes by the laser?

Answer 14

One that is directly proportional to the amount of fluorescent probe that is conjugated to the cell

Question 15

What happens after the laser beam strikes the cell?

Answer 15

Light is scattered in all directions, and light emission subsequently enables the measurement of several biochemical, biophysical, and molecular properties

Question 16

What happens in the optical and electronics system?

Answer 16

It detects light that is scattered forward, light that is scattered at 90 degrees, and the fluorescent light signals that are emitted upon excitation

Question 17

What does the data storage and computer control system do?

Answer 17

Interprets the recorded signals and presents them as comprehensible data

Question 18

What does a flow cytometer ultimately do?

Answer 18

Analyzes the light scattering parameters and generate data related to the structural and morphological features of a cell

Question 19

What is the intensity of the low angle forward scatter proportional to?

Answer 19

Cellular diameter and size

Question 20

What does the orthogonal or side (90 degree) intensity indicate?

Answer 20

The quantity of granular structures within the cellular population

Question 21

What else is light scatter alone often quite useful and commonly used for?

Answer 21

Excluding dead cells, cell aggregates, and cellular debris from the generated fluorescence data

Question 22

How will flow cytometry be used in this lab?

Answer 22

As a means to examine the cell cycle

Question 23

What is the protocol for this lab based on?

Answer 23

The fact that at different stages of the cell cycle, cells have different DNA content

Question 24

What kind of dye is used in this lab?

Answer 24

One that allows quantitation of the amount of cellular DNA - Propidium iodide (PI)

Question 25

What can PI be used to stain?

Answer 25

Whole cells or isolated nuclei

Question 26

How does PI work?

Answer 26

It intercalates into the major groove of double-stranded DNA and produces a highly fluorescent adduct that can be excited at 488 nm with a broad emission centered around 600 nm

Question 27

Why will we utilize both forward and side scatter to classify populations of cells?

Answer 27

Since cells vary in size and shape at different stages of the cell cycle

Question 28

What will be determined in the original flow cytometry analysis?

Answer 28

The effect of serum starvation, nocodazole or aphidicolin treatment on cell cycle profiles

Question 29

What is a key approach in cell cycle analysis and cancer research?

Answer 29

Studying the phenotypes that are present in a cell when important proteins are either overexpressed or deleted

Question 30

What will also be monitored in this lab?

Answer 30

The effect of overexpression of a transgene fused to a GFP reporter

Question 31

What is done to the transgene that is transiently transfected into HEK 293 cells?

Answer 31

GFP expression is used to distinguish between cells that were successfully transfected and those that were not to therefore determine what effect, if any, overexpression of the transgene has on the cell cycle in the transfected cells

Question 32

What is some important information pertaining to this lab?

Answer 32

• Working with live cells
• Perform steps at room temperature
• Cells can be lysed and destroyed easily
• If cells are not fixed properly, fragmentation of DNA will be detected during flow cytometry analysis