Flow Cytometry

Question 1

What is flow cytometery

Answer 1

Powerful technique used to analyse different properties of cell population
E.g. human blood cells

Question 2

Why use blood cells

Answer 2

Their absolute numbers and relative abundances change in different disease states
They have suspension
They have different sizes (forward scatter)
They have different granularities (side scatter)
They express proteins on their surface that can be lineage specific

Question 3

How do we visuales and count cells within blood samples by flow cytometry

Answer 3

Separating different cell subsets by size (FSC) and/or granularity (SSC)

Question 4

How are cells analysed

Answer 4

One at a time for these properties while in suspension as they ‘flow’ through the instrument

Question 5

What do cells pass through to be analysed

Answer 5

A laser beam where they scatter the laser light and emit florescence which is collected by special detectors. These detectors convert the photons into a voltage pulse

Question 6

How many cells pass through the machine every second

Answer 6

Thousands and their properties are evaluated and recorded

Question 7

Fluidics and what enable cells analysed individually

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Answer 7

Optics

Question 8

How do cells generate voltage pulse

Answer 8

When there is no cell in the beam there is no signal

As a cell enters the beam the voltage pulse increases

When the whole cell is in the beam the pulse reaches its peak

As the cell leaves the Beam the pulse comes down

When the cell is no longer in the beam there is no signal

Question 9

Raw data captured as individual cells pass through

Answer 9

Cells with high fluorescents cell signals have higher peaks than low flourescents