HTHSCI 1DT3 Lecture Notes - Lecture 7: Apoptosis, B Cell, Paraformaldehyde
23 Jun 2018
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Culturing Secondary Cells:
Secondary cultures are cultures composed of the progeny of primary cells that have been allowed to
divide in culture.
Expanded secondary cultures provide large numbers of mixed or separated cell types for cellular
and biochemical analysis.
However, the process of cell senescence means that primary cells will only divide a finite
number of times (e.g. 25-40 for human fibroblasts)
Therefore, immortalised cell lines are very commonly used in molecular and cellular
neurobiology research.
These divide an infinite number of times, but still obey culture conditions (e.g. ceasing division
when they reach confluency)
Common Cell Lines:
3T3 (Swiss & NIH) – mouse fibroblast
PC12 (phaeochromocytoma) – rat chromaffin
COS – monkey kidney
CHO – chinese hamster ovary
MDCK – dog epithelial
HeLa – human epithelial
L6 – rat myoblast
293 – human kidney (transformed)
Of these, only HeLA cells grow in suspension, the rest require solid substrata.
Stimulation of Survival and Proliferation by FCS
The most common cell culture is DMEM containing 10% FCS
This is the standard medium for culturing most cell types, such as fibroblasts, epithelial cells
and astrocytes.
DMEM/10% FCS stimulates the cells to survive and also to proliferate, usually doubling every
24h on average.
At lower FCS concentrations (e.g. DMEM/FCS 0.5%) the cells can survive, but will proliferate
much more slowly, if at all.
For routine culture, where you want cells to proliferate, 10% FCS is therefore standard.
However, if you are testing the proliferative effect of a growth factor, you might use lower FCS
concentrations, such as 0.5%, which keeps the cells alive, but not dividing.
In Vitro Assays
Assays for Proliferation:
DNA Synthesis Assays:
Before cells divide, they need to replicate all their DNA (during the S-phase of the cell
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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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Document Summary
Secondary cultures are cultures composed of the progeny of primary cells that have been allowed to divide in culture. Expanded secondary cultures provide large numbers of mixed or separated cell types for cellular and biochemical analysis. However, the process of cell senescence means that primary cells will only divide a finite number of times (e. g. 25-40 for human fibroblasts) Therefore, immortalised cell lines are very commonly used in molecular and cellular neurobiology research. These divide an infinite number of times, but still obey culture conditions (e. g. ceasing division when they reach confluency) Of these, only hela cells grow in suspension, the rest require solid substrata. The most common cell culture is dmem containing 10% fcs. This is the standard medium for culturing most cell types, such as fibroblasts, epithelial cells and astrocytes. Dmem/10% fcs stimulates the cells to survive and also to proliferate, usually doubling every.
