8 | Flow Cytometry (incl. FACS stability analysis)

Question 1

What four methods can be used to analyse stability?

Answer 1

FACS
CPM
CETSA
CD

Question 2

When we talk about GPCR screening, what 3 methods are there to analyse receptor binding?

Answer 2

FRET
SPR
ITC

Question 3

When we talk about transport, what 3 whole-cell assays are there wrt. aquaporins

Answer 3

Epithelial cell layer
Suspended cell or vesicle
Erythrocyte suspension

Question 4

When we talk about transport, what 3 osmotic pressure assays are there wrt. aquaporins

Answer 4

Sphereplast
Proteoliposomes
‘Biacore’

Question 5

What does the Hour-Glass model explain?

Answer 5

it predicts functional structure from mutational analysis of constriction regions, which explains the specificity at a certain pore diameter for water wrt. selectivity filter

Question 6

When we talk about protein interactions (PPI’s), name one conventional method to identify these interactions, and one fluorescent metgod?

Answer 6

Yeast-two-hybrid interaction assay
FRET

Question 7

What does flow cytometry allow for?

Answer 7

detailed characterisation of distinct populations within heterogeneous pictures of particles

Question 8

Flow cytometers allow for investigating up to 20 fluorescence parameters based on derived particle properties, as well as using scattering of light to resolve to resolve certain cell populations based on their?

Answer 8

size and internal complexity

Question 9

for FACS, in terms of construction which three main components are there?

Answer 9

Fluidics system
Optical system
Signal detection and processing system

Question 10

Fluidics system:
What two components are included?

Answer 10

Central core: SIP, sample line
Outer sheath fluid: creates a single stream of particles to be characterised individually, by using a higher pressure than sample under laminar flow – causing a drag effect which align particles in a single file, i.e., hydrodynamic focussing before reaching ‘interrogation point’

Question 11

Fluidics system:
By manipulating the flow rate of cells passing the laser beam, what strength of flow should be used for immunophenotyping cells, and for assessing DNA content of a cell?

Answer 11

High
Low

Question 12

Optical system:
What 2 types of scattered light is analysed?
Explain each of them!

Answer 12

Forward scatter (FSC):
Diffracted/refracted light collected along the same direction as the incident laser beam (and up to about 20° offset from that direction). The intensity is proportional to area of cell surface –> estimation of cell size.
________________
Side scatter (SSC):
Measures reflected light giving a assessment of internal complexity

Question 13

Optical system:
what does both FSC and SSC properties give information about, writ. differentiation?

Answer 13

specific cell types within a heterogeneous
cell population such as the blood.

Question 14

Signal detection and processing system:
What two different types of detectors are commonly used in flow cytometers?

Answer 14

1. PD’s:
   greater efficiency of light to photoelectron conversion)
2. PMT’s:
   Greater sensitivity

Question 15

The general principles of FACS are the same as those for analytical flow cytometry
in that cells are assessed based on
fluorescence characteristics of the mixed cell population, either by ?

Answer 15

1. binding of fluorescently conjugated antibodies
2. or genetic encoding/expression of a
   fluorescent protein.

Question 16

In FACS, explain gating

Answer 16

it allows sorting by electrostatic charge under droplet formation using a electrostatic deflection system– in which we’re able to separate cells expressing different markers (and therefore bound by different
fluorescently conjugated antibodies)

Question 17

Perhaps the most common, and indeed most famous, use for flow cytometry is in
immunophenotyping. This is used by research scientists to ?

Answer 17

define populations of
cells