Cell Biological Techniques 1 Flashcards
What are primary cells?
isolated from tissues ex vivo model difficult to isolate onyl grow for limited no. of generations difficult to manipulate genetically
What are cell ines?
derived from tumours can grow for many generations ease of culture easily transfected make sure accurate reflection of properties of cells found in vivo may not replicate all cell features
What are HeLa cells?
cervical carcinoma cell line
transformed by HPV18
immortalised
v easy to culture & transfect
What are iPS cells?
reprogrammed adult somatic cells
isolate fibroblasts & transduce with transcription factors - turn cells into stem cells
promote differentiation
mimic primary cells
How are mammalian cells cultured?
grown in humidified incubator at 37c
grow in suspension of adhere to coated plastic surface
manipulations performed in lamina flow hood - prevent contamination
What is growth media typically supplemented with?
serum derived from foetal calves (FCS)
Why transfect cells with nucleic acids?
POI may not be expressed in cells to be studied
May be no antibody to detect the protein
may want to study mutant form
What are mammalian expression constructs?
DNA plasmids that can be propagated in E.coli - have selection markers
gene is inserted downstream of a promoter
Tags added to coding sequence for detection e..g GFP or epitope tag
How is RNA interference (RNAi) used to knockdown gene expression?
uses siRNAs to knockdwon gene expression
antisense strand of siRNA bound by RISC - cleaves mRNA with complementary sequence preventing tranlsation
What are the 4 ways mammalian cells can be transfected?
Electroporation
Lipid based reagents
Calcium phosphate
Injection of nucleic acids
what does transfection do?
introduced the DNA construct of siRNA into the cells
What is the resolution of light microscopy?
cells <0.2um apart
additional info added by fluorescent proteins, antibodies and dyes
How does immunofluorescence microscopy work
detects proteins with antibodies specific for the protein/epitope tags
expression and localisation of more than 1 protein can be analysed in same sample
using antibodies raised in diff. species
see if they colocalise
How does Electron microscopy work?
preparation - chemical fixation, dehydration, sectioning & embedding
resolution is 200X greater
sections can be stained with antibodies labelled with gold
How does cryo-electron tomography (cryo-ET) work?
near-atomic resolution
samples frozen and sectioned thinly
stage tilts
3D image
What is subcellular fractionation?
separates subcellular organelles by differential centrifugation
What are some applications of subcellular fractionation?
identify where protein is in cell
identify proteins associated with organelle
analysis of processes associated with specific organelle
What are the stages of differential centrifugation?
cells homogenised
homogenate spun at low speed - separate nuclei
high speed centrifugation - separate other organelles
What are the stages of density gradient centrifugation?
cells homogenised
postnuclear supernatant generated by centrifugation
post nuclear supernatant loaded onto density gradient (dense ionic substance e.g. sucrose/sugars)
organelles reach buoyant density
What is an example of density gradient fractionation?
YTS natural killer line - isolate secretory lysosomes
What is an exampe of differential centrifugation?
HeLA cells
analysis of fractions by immunoblotting
