Methods in cell biology Flashcards
what is the issue of using cells from animal tissue or individuals?
they can vary from sample to sample and are genetically diverse lacking consistency
what happens to cells removed from their natural environment?
can become stressed and die without proper nutrients
how can you keep isolated tissue alive?
simple salt solutions should keep them for a few days
when do cultured cells die?
after a few generations ( 40-60)
how do telomeres affect cell grwoth?
telomeres shorten following cell division and limit the number of times a cell a divide
how do tumour cells become immortal?
they can rebuild their temomeres so become immortal
where did HeLa cell orginiate?
henrietta Lacks 1952 cervial tumour
why did HeLa cells become a thing?
they were immortal and could be cultured indefinately
made into a cel line
how can we now make immortal cell lines?
infect them with SV40 which activated telomerase so its a normal cell with an immortal function
what do typical cell culture medias contain?
salts amino acids vitamins such as folic acid and riboflavin glucose re phenol dye buffers
what does confluent mean?
cels run out of space in the dish and cover alll the space at the bottom
signals they need to be split into new dishes and replated
how are cells moved to a new dish?
an enzyme trypsin detaches them
they are passaged and allow to form new connections on the disk
are HeLa cells adhernent or not?
they are adherent
what does passaging mean?
its the process of allowing cells to make new attachements
needs to happen 1-2 times a week
what is a non-adhernt cell?
blood cells
require dilute medials in suspension
name some basic cell culture pricnciples
aseptic technique
kept in sterile conditions
no contimination
who made the first pimitive micrscope?
Robert Hooke 1665
what did the Van Leeuwenhoek microscope look like?
very advanced for the time
used to discover sperm, red bood cels, protists and muscle fibres
what are the three types of light microscope around today?
bright field
phase contrast
differential interference contract DIC
what is H&E stain
most common staining method
what does haemoatoxylin do?
stain acid strcutres such as nucleus
Purpleblue
what does eosin do?
stain basic strctures such as cytoplasm
pink
what colours does the azan trichrome stain use?
nuclei red
collagen/BM blue
muscule and RBC orange
used for connective tissues and epitheliums
when were the stages of mitosis discovered?
1878 by Walher
how was mitosis discovered?
dyes such as aniline used to visualise chromosomes
what is aniline?
specialise DNA stain
how do fluorescent microscopes work?
shine light at one wavelength at the cells and it reflects it at different wavelength
filters are used too
how are antibbodies ued in fluorescent microscopes?
they can recognise a specific point or thing in the target molecule so you can see this certain thing
they bind to biological molecules
secondary antibodies can then bind with fluorescent markers for colours which amplify the signal
how is the location of the fluorescence detected in the tissue?
microscopes
why is live cell imaging used?
cells are dynamic and processes occur over time
want to visualise these processes rather than a snapshot of them
how does live cell imaging work?
uses an inverted microscope
objective is placed under the micscope allowing view of cells in culture media
how are cells kept alive in live cell images?
environment chamber maintains the temp, adjusts gas suply to maintain pH
kept alive and health for several days under the microscope
what is GFP
a gene from a jellyfish
what was the original form of GFP like?
worked best at sea temps and onyl weakly fluoreced, easyily bleached and unstable
what is gfp like now?
mutant version workes at 37degrees and can now work as a monomer and is more photostable
what are the uses of gfp?
track embryo development
protein dynamics
what develoments are there for gfp now?
multi colours of it now can be used to track multiple things
eg brain neurones all together
what is FRAP
fluorescence recovery after photobleaching
when is FRAP used?
determine kinetics of protein redistrubution and detect how much of the proteinis immobile or membrane bound
what other types of gfp are there?
RFP
isolated from coral
what is intravital imagining?
the use of GFP in cells or tissues in live organisms being viewed
what is photo activation
laser shined onto tissues and changed colour after stimulation
allows tracking of sub-population cells
what arethe main limitations of photoactivation?
- photobleaching
- phototoxicity
- overexpression
- protein folding
- cellular distrubution
what is photobleaching?
limitation of photoactivation
fluorescent proteins are sensitive to bleaching so long term studies can be tricky
what is photo-toxicity
limitation of photoactivation
illumination of cells with high-intensity light can be toxic formin free radicals
what is overexpression?
limitation of photoactivation
tagged genes are often expressed at high levels to make a good signal
excess proteins can after the cells normal distrubution
what is cellular distribution in relation to photoactivation?
the large fluorescent proteins may affect the cellular disrubution of the tagged proteins which is especially key for membrane proteins
what is a confocal microscope?
modified version of fluorescent miscopscopy
how does a confocal microscope work?
lasers of specific wavelength are used to illuminate molecules in the sample
3D image of the cell with cross sections
advantages of a confocal microscope
- 3D view
- cross sections
- info on spatial relationship
- used on both cells and tissues
advantages of TEM
highest resolution possible
how does TEM work?
antibodies are labelled with gold to detect cellular structures
the disadvantage of TEM
requires very thin sections of cells
what is super resolution microscopy?
provides 3D view of cells within a tissue and looks at spatial relationships within the tissue
can super resolution or confocal do timelapses?
no
laser light is scattered by skin and other tissues and does not penetrate much
what is STORM?
stochastic optical resolution
what does STORM do?
allows you to determines the exact centre of the fluorescent signal and therefore the exact location o the dye molecule
limitations to STORM
can be very slow
photobleaching and toxicity can be an issue
limited use of GFP
expensive
