Culture and analysis of Mammalian Cells Flashcards
What are primary cells?
Isolated from tissues
An ex vivo model to study cells
Can be difficult to isolate and in limited supply e.g. human neurons
If they can divide in vitro (cell culture lab) they will only grow for a limited number of generations and they can de-differentiate
Can be difficult to manipulate genetically e.g. transfection of genes
Example - isolation of primary human natural killer cells from blood
What are cell lines?
A way to study cells in the lab
Typically derived from tumours
Can grow in the lab for many generations if not indefinitely
Abnormal when compared to primary cells - as they are tumour derived
Commonly used in research due to ease of culture and their ready availability
Typically more easily transfected with nucleic acids than primary cells
May retain some, but not usually not all features, of cells from which tumour is derived
Give an example of a well known cell line?
HeLa cells
A cervical carcinoma cell line derived from an aggressive tumour
Transformed by human papilloma virus 18 and these cells have an abnormal number of chromosomes
Immortalised and can be grown indefinitely
Used throughout the world as a model human cell line in research
Easy to culture, transfect and use in cell biological experiments
What are induced pluripotent stem cells?
iPS cells are reprogrammed adult somatic cells
Isolate fibroblasts and transduce these with transcription factors that turn the cells into stem cells that have the same properties as embryonic stem cells
Additional factors including culture conditions can then be used to promote differentiation into defined cell populations
Used to mimic primary cells and can be isolated from subjects with genetic diseases
What is the mechanism of culturing mammalian cells?
Cells are typically grown in plastic flasks/dishes in a humidified incubator at 37C
Cells can either grow in suspension (e.g. lymphocytes) or adhere to the specially coated plastic surface (e.g. epithelial cells)
Performed using sterile media and materials to avoid microbial contamination
All manipulations are performed in a laminar flow hood to prevent airborne contamination
Describe culture media used in culturing mammalian cells?
Cells are grown in growth medium that contains nutrients and salts that provide optimal conditions for cell growth
Growth medium is typically supplemented with serum, usually derived from foetal calves (FCS)
This provides growth factors required for many cell types
Additional factors such as cytokines can be added to promote the growth and differentiation of certain cell types
Why do we transfect cells?
The protein of interest may not be expressed in the cells to be studied e.g. a pathogen protein
There may be no antibody to detect the protein: express a tagged form of the protein
You may want to study a mutant form of a protein associated with disease
You may want to analyse mutants in order to define sequences of the protein that are important for its function
What is a mammalian expression construct for transfection?
Many mammalian expression constructs are DNA plasmids that can be propagated in E. coli and have selection markers for this e.g. AmpR
The gene (X) is inserted downstream of a promoter that functions in mammalian cells
Tags are often added to the mammalian gene’s coding sequence to enable detection of the expressed protein
Fluorescent proteins (GFP, RFP)
Epitope tags that can be detected with antibodies eg HA, FLAG, Myc
What is interference RNA (RNAi) used for?
This is used to knockdown gene expression in order to test is the protein encoded by this gene is important for a cellular function/pathway
siRNA is used to knockdown gene expression in a sequence specific fashion
Small double stranded siRNAs specific for the target gene are transfected into mammalian cells
The antisense strand of the siRNA is bound by an RNA-induced silencing complex (RISC), which cleaves mRNAs with a complementary sequence preventing them from being translated
What is the mechanism of transfection of mammalian cells?
Transfection introduces the DNA construct or siRNA into the cells
Cells can be transfected by a variety of different methods.
The method chosen is cell type dependent and include:
Electroporation: electrical field increases permeability of the membrane to DNA/siRNA to cross the membrane into the cell
Lipid based reagents: encapsulate DNA/siRNA and fuse with the membranes of the cell delivering the DNA to the cytosol
Calcium phosphate: forms precipitate with DNA that is taken up by cells
Injection of nucleic acids
How can we observe cells?
Immunofluorescence microscopy
Electron microscopy
Cryo-electron microscopy
Describe microscopy of cells?
Mammalian cells are typically 10-20 mm in diameter, colourless and translucent
Cells can however be observed with microscopes that use visible and UV light or electrons
Light microscopy can resolve parts of a specimen that are <0.2mm apart
Light microscopy can be used to image cells, but additional information is provided by using fluorescent proteins, antibodies and dyes
Describe immunofluorescence microscopy?
Detects proteins with antibodies specific for the protein or epitope tags
Expression and localisation of more than one protein can be analysed by in same sample by using antibodies raised in different species
The relative distribution of proteins to be analysed e.g. do they co-localise in the same organelle
A rabbit anti-X antibody in combination with a mouse anti-Y antibody
X can then be visualised with a fluorescently labelled secondary antibody specific for rabbit immunoglobulins (Igs) and Y with a fluorescently labelled secondary antibody specific for mouse Igs.
The secondary antibodies are labelled with different fluorescent dyes
They emit light at different wavelengths
What are fluorescent proteins?
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria is most commonly used
There is now a spectrum of different fluorescent proteins that when excited fluoresce at different wavelengths (colours)
GFP emits green light (509 nm) when excited with light at 395nm, enabling direct visualisation in a cell
Fluorescent proteins can be imaged in live cells enables not only the localisation of proteins to be monitored but also their movement
Describe electron microscopy?
Sample preparation for EM can be complex and can involve chemical fixation, dehydration, sectioning and embedding
However, the resolution of EM is 200X greater than light microscopy, enabling cellular structures to be resolved in fine detail i.e. ultra structure
Sections can be stained with antibodies labelled with gold particles
