Chapter 8: Flow cytometry

Question 1

Flow cytometry is a technique that allows for:

How is it achieved?

Answer 1

rapid measurement of optical and fluorescence properties of large numbers of individual particles

stream of particles + multiple light beams

Question 2

In flow cytometry, multiple features of each particle

can be investigated simultaneously, what does this allow for?

Answer 2

characterization of distinct populations within heterogeneous mixtures of particles –> detailed investigation

Question 3

Is flow cytometry the most common use for the sample type: cells?

Answer 3

yes

Question 4

Describe FACS!

Answer 4

capability to sort specific particles based on the fluorescence properties interrogated, thereby allow for particles with desired parameters to be isolated

Question 5

with which type of technology can flow cytometry be combined with to explore molecular heterogeneity?

Answer 5

high-throughput sequencing technologies

Question 6

In terms of construction, flow cytometers comprise three main components, which three?

Answer 6

a) Fluidics system with SIP –> sheath fluid (hydrodynamic focussing) –> central core –> single stream of cells
b) Optical system –> interrogation point (to laser beam via lenses and optics) –> light scattering by FSC & SSC
c) Signal detection (PDs or PMTs) –> current –> amplifier –> digital signal by ADC and processing system

Question 7

regarding the amplifiers, what use do we have for linear or logaritmic?

Answer 7

1. linear:
   DNA analysis
   2: logarithmic
   fluorescence studies of immune cell populations

Question 8

FLUORESCENCE-ACTIVATED CELL SORT

flow cytometry can also be used to in addition to qualitatively and quantitatively assess cell phenotype
and function?

Answer 8

separate particles or cells based on fluorescence labelling with conjugated antibodies to 95-98% purity

then the cells are gated to identify the cell population of intrest to be sorted

Question 9

FACS

why would we use fluorescence labels?

Answer 9

to study cell anatomy, cells need to be stained to prospect biological components

Question 10

FACS

which is the most common fluorescent dye?

Answer 10

FITC

Question 11

FACS

why would we use fluorescently labelled toxins?

Answer 11

due to sensitivity making good markers

Question 12

FACS

name one fluorescence protein label

Answer 12

GFP

Question 13

FACS

why would one use antibodies as labels?

Answer 13

For specific labelling of cell populations

Question 14

What is the most famous and common use for flow cytometry?

Answer 14

immunophenotyping; to define populations of

cells