HTHSCI 1DT3 Lecture Notes - Lecture 8: Oligodendrocyte, Fragment Crystallizable Region, Neuroglia
23 Jun 2018
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Before cells divide, they need to replicate all their DNA (during the S-phase of the cell
cycle/synthesis phase)
If labelled nucleotides are added to the culture medium they will incorporate into the
chromosomes of dividing cells.
This provides a means of scoring the proportion of cells that have entered S phase,
within a given culture period.
The most common nucleotide labelling is either titrated thymidine, which can be detected
because it is radioactive, or bromodeoxyuridine (BrdU), which can be detected by
immunocytochemistry using a specific anti-BrdU antibody.
Assaying for increases in cell number:
Colorimetric assay: Addition of a tetrazolium salt (MTT or MTS) that changes colour
from bright yellow to brown, when reduced by electrons from the electron transport
chain in respiring cells.
(other ways of obtaining quantitative data on cell number: Manual counting using a
microscope, either in situ or after trypsininsation, or FACS)
Assaying for apoptosis:
TUNEL Assay: (Nick end labelling)
Double-strand nicks are symptomatic of DNA of cells undergoing apoptosis.
Addition of DNA polymerase I together with fluorescently-tagged nucleotides
allows labelling of nicked DNA.
The nuclei of cells undergoing apoptosis shows up clearly under the
fluorescence microscope.
By staining total nuclei e.g. using propidium iodide, the proportion of cells
undergoing apoptosis can be determined.
DNA Laddering
This is a biochemical means of assaying apoptosis.
Cells dying by apoptosis cleave their DNA into regular lengths.
Isolated DNA from apoptotic cells appear as a ladder of DNA fragments.
Cell Number (Survival) Assay
This employs the same MTT/MTS reagent for the proliferation assay.
What distinguishes the two is whether the cell number has risen from the starting
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What distinguishes the two is whether the cell number has risen from the starting
number (proliferation) or dropped (Apoptosis)
The degree of ‘brown-ness’ assayed at 450nm is proportional to the number of
respiring cells.
There are also more specific apoptotic markers that can be assessed (specific to different
apoptotic pathways) e.g. detecting the products of the regulated protein cleavage that is
characteristic of apoptosis (e.g. cleavage of PARP).
In general, at least two different assays for apoptosis are required to convince that cells are
dying by apoptosis e.g. MTS+TUNEL
Neuronal Culture
Neurons require culture on an adherent substratum coated with poly-L-lysine, collagen
or laminin
They can either be cultured in mixed culture with other cells, or purified.
In mixed cultures, glial cells soon proliferate profusely even if outnumbered by neurons
on plating (because neurons can’t divide).
In long term neuronal cultures, the proliferation of non-neural cells can be inhibited
using mitogenic inhibitors which kill the dividing cells, but do not affect post-mitotic
neurons.
However – in many cases, neurons thrive in culture in the presence of glial cells e.g.
motor neurons culture.
For some assays, the presence of glial cells is required.
E.g. to study the mechanisms underlying myelination and remyelination it would
be useful to have oligodendrocyte-neurone co-culture in which the
oligodendrocytes could myelinate the neurons as physiologically as possible.
Astrocytes enhance synapse formation.
Important if biochemistry relating to synapses is being studied in the neuronal
cultures.
Unlike dividing cells, it is difficult to introduce transgenes into neurons in culture.
Using calcium phosphate precipitation, electroporation or lipofectamine-based
methods, efficiencies are very low
Retroviral vectors are no use as they require the target cell to be dividing.
Other viral vector systems are promising (e.g. herpes virus, lenti-virus,
adenovirus based vectors, but require a large investment of time to set up)
For this reason, to test the effect of aberrant protein expression on neuronal
morphology or behaviour, researchers turned to neuronal cell lines.
The PC12 cell line is a rat chromaffin cell from a rad adrenal glad tumour. It is
an immortal cell line that divides infinitely in culture.
When grown in the presence of NGF, it differentiates into a sympathetic
neuronal morphology.
Mature PC12 cultures acquire a cholinergic phenotype and form functional
autapses (synapses that innervate the neuron itself)
Therefore it has the advantage that it can divide in culture, and transgenes can be
introduced into it and inherited to create a large population of transgene of
transgene-expressing cells. But can also be chemically changed into a neuron to
be used to study the effect of the introduced protein on neuronal responses.
These cells have been extensively used to explore signalling pathways associated
with the effects of NGF on neurons.
Caution is required in interpreting these results. It must be remembered that
PC12 is a cell line, and as such its growth factor signalling pathways are
unlikely to represent the signalling in a primary sympathetic neuron.
Nevertheless they are very useful for determining the effects of expressing
certain proteins involved in differentiation or survival of neurons.
There are other neuroblastoma cell lines that are commonly used also (e.g.
Neuro2A N2A)
Other considerations for cellular experiments:
The secret of a good cell culture assay is to make it as physiologically relevant as
possible, but simple and well-controlled to get clear answers.
E.g. research into how binding of a protein on the surface of neurons (neural cell
adhesion molecule – NCAM) to the same protein on the surface of other cell
changes the elongation of neurons.
Originally, researchers used primary cultures of neurons plated onto cell culture
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Originally, researchers used primary cultures of neurons plated onto cell culture
plates that had been coated with purified NCAM proteins (immobilised proteins)
However, we need to ensure we are studying the interaction as it would occur in
tissue, so we therefore express NCAM in a cell line that does not express it, and
look at its effect on neurons compared to the effect of non-NCAM transfected
cells.
Thus NCAM is being presented to the neuron in a physiologically relevant
assay, but we can assign any effect specifically to NCAM on the neuron
interacting with NCAM on the surface of the cellular monolayer.
Other examples of approaches used to study neuronal responses:
Explant Cultures:
Have been extensively used to determine the tropic, trophic and neuritogenic effects of
various proteins involved in nervous system development.
In a typical experiment, pieces of tissue are suspended in a semi-solid matrix such as
collagen gel, a small distance from one another. The influence of one on the other can be
determined.
For example, if a piece of dorsal spinal cord is placed in a collagen gel with a piece of
floor plate tissue, neurons from the dorsal tissue will grow towards the floor plate
tissue. This is because the floor plate secretes netrin which is attractive to commissural
neurons in the dorsal tissue.
The observation of activities such as this led to the purification of guidance molecules
such as netrin, semaphorins, etc. Pieces from pellets of cell lines transfected with a
particular potential guidance molecule can be tested in this assay for their effects on
target tissue.
Immunocytochemistry
Cells are transparent and difficult to see by light microscopy.
Histological dyes such as hematoxylin and
eosin (H&E) allow subcellular structures
to be identified.
However, use of antibodies
(immunocytochemistry) provided a
revolutionary ability to identify specific
sub-cellular structures (e.g. microtubules
can be identified specifically using a
tubulin-specific antibody) – right.
Confocal Fluorescent Microscopy
Allows a single optical section to be seen, as the out-of-focus light that reduces the definition.
The image is processed by computer.
This is important when working with thick tissue samples, but less important for visualising
components in cells in culture, which are relatively flat.
Fluorescent microscopes that are not confocal are known as wide-field microscopes.
Revision
Antibodies:
Produced in the blood by B-lymphocytes.
For any foreign material (immunogen or antigen) that may enter the blood there is a B-
lymphocyte that will recognise it.
Binding to a foreign target causes that B-lymphocyte to divide to produce a large clonal
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Document Summary
Before cells divide, they need to replicate all their dna (during the s-phase of the cell cycle/synthesis phase) If labelled nucleotides are added to the culture medium they will incorporate into the chromosomes of dividing cells. This provides a means of scoring the proportion of cells that have entered s phase, within a given culture period. The most common nucleotide labelling is either titrated thymidine, which can be detected because it is radioactive, or bromodeoxyuridine (brdu), which can be detected by immunocytochemistry using a specific anti-brdu antibody. Assaying for increases in cell number: o o. Tunel assay: (nick end labelling) i. ii. iii. iv. Double-strand nicks are symptomatic of dna of cells undergoing apoptosis. Addition of dna polymerase i together with fluorescently-tagged nucleotides allows labelling of nicked dna. The nuclei of cells undergoing apoptosis shows up clearly under the fluorescence microscope. By staining total nuclei e. g. using propidium iodide, the proportion of cells undergoing apoptosis can be determined. o.
