Cell Biological Techniques 1 Flashcards

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1
Q

What are primary cells?

A
isolated from tissues
ex vivo model 
difficult to isolate
onyl grow for limited no. of generations
difficult to manipulate genetically
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2
Q

What are cell ines?

A
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
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3
Q

What are HeLa cells?

A

cervical carcinoma cell line
transformed by HPV18
immortalised
v easy to culture & transfect

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4
Q

What are iPS cells?

A

reprogrammed adult somatic cells
isolate fibroblasts & transduce with transcription factors - turn cells into stem cells
promote differentiation
mimic primary cells

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5
Q

How are mammalian cells cultured?

A

grown in humidified incubator at 37c

grow in suspension of adhere to coated plastic surface

manipulations performed in lamina flow hood - prevent contamination

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6
Q

What is growth media typically supplemented with?

A

serum derived from foetal calves (FCS)

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7
Q

Why transfect cells with nucleic acids?

A

POI may not be expressed in cells to be studied

May be no antibody to detect the protein

may want to study mutant form

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8
Q

What are mammalian expression constructs?

A

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

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9
Q

How is RNA interference (RNAi) used to knockdown gene expression?

A

uses siRNAs to knockdwon gene expression

antisense strand of siRNA bound by RISC - cleaves mRNA with complementary sequence preventing tranlsation

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10
Q

What are the 4 ways mammalian cells can be transfected?

A

Electroporation
Lipid based reagents
Calcium phosphate
Injection of nucleic acids

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11
Q

what does transfection do?

A

introduced the DNA construct of siRNA into the cells

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12
Q

What is the resolution of light microscopy?

A

cells <0.2um apart

additional info added by fluorescent proteins, antibodies and dyes

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13
Q

How does immunofluorescence microscopy work

A

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

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14
Q

How does Electron microscopy work?

A

preparation - chemical fixation, dehydration, sectioning & embedding

resolution is 200X greater

sections can be stained with antibodies labelled with gold

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15
Q

How does cryo-electron tomography (cryo-ET) work?

A

near-atomic resolution
samples frozen and sectioned thinly
stage tilts
3D image

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16
Q

What is subcellular fractionation?

A

separates subcellular organelles by differential centrifugation

17
Q

What are some applications of subcellular fractionation?

A

identify where protein is in cell

identify proteins associated with organelle

analysis of processes associated with specific organelle

18
Q

What are the stages of differential centrifugation?

A

cells homogenised
homogenate spun at low speed - separate nuclei
high speed centrifugation - separate other organelles

19
Q

What are the stages of density gradient centrifugation?

A

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

20
Q

What is an example of density gradient fractionation?

A

YTS natural killer line - isolate secretory lysosomes

21
Q

What is an exampe of differential centrifugation?

A

HeLA cells

analysis of fractions by immunoblotting