Methods in cell biology

Question 1

what is the issue of using cells from animal tissue or individuals?

Answer 1

they can vary from sample to sample and are genetically diverse lacking consistency

Question 2

what happens to cells removed from their natural environment?

Answer 2

can become stressed and die without proper nutrients

Question 3

how can you keep isolated tissue alive?

Answer 3

simple salt solutions should keep them for a few days

Question 4

when do cultured cells die?

Answer 4

after a few generations ( 40-60)

Question 5

how do telomeres affect cell grwoth?

Answer 5

telomeres shorten following cell division and limit the number of times a cell a divide

Question 6

how do tumour cells become immortal?

Answer 6

they can rebuild their temomeres so become immortal

Question 7

where did HeLa cell orginiate?

Answer 7

henrietta Lacks 1952 cervial tumour

Question 8

why did HeLa cells become a thing?

Answer 8

they were immortal and could be cultured indefinately

made into a cel line

Question 9

how can we now make immortal cell lines?

Answer 9

infect them with SV40 which activated telomerase so its a normal cell with an immortal function

Question 10

what do typical cell culture medias contain?

Answer 10

salts 
amino acids
vitamins such as folic acid and riboflavin
glucose
re phenol dye
buffers

Question 11

what does confluent mean?

Answer 11

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

Question 12

how are cells moved to a new dish?

Answer 12

an enzyme trypsin detaches them

they are passaged and allow to form new connections on the disk

Question 13

are HeLa cells adhernent or not?

Answer 13

they are adherent

Question 14

what does passaging mean?

Answer 14

its the process of allowing cells to make new attachements

needs to happen 1-2 times a week

Question 15

what is a non-adhernt cell?

Answer 15

blood cells

require dilute medials in suspension

Question 16

name some basic cell culture pricnciples

Answer 16

aseptic technique
kept in sterile conditions
no contimination

Question 17

who made the first pimitive micrscope?

Answer 17

Robert Hooke 1665

Question 18

what did the Van Leeuwenhoek microscope look like?

Answer 18

very advanced for the time

used to discover sperm, red bood cels, protists and muscle fibres

Question 19

what are the three types of light microscope around today?

Answer 19

bright field
phase contrast
differential interference contract DIC

Question 20

what is H&E stain

Answer 20

most common staining method

Question 21

what does haemoatoxylin do?

Answer 21

stain acid strcutres such as nucleus

Purpleblue

Question 22

what does eosin do?

Answer 22

stain basic strctures such as cytoplasm

pink

Question 23

what colours does the azan trichrome stain use?

Answer 23

nuclei red
collagen/BM blue
muscule and RBC orange
used for connective tissues and epitheliums

Question 24

when were the stages of mitosis discovered?

Answer 24

1878 by Walher

Question 25

how was mitosis discovered?

Answer 25

dyes such as aniline used to visualise chromosomes

Question 26

what is aniline?

Answer 26

specialise DNA stain

Question 27

how do fluorescent microscopes work?

Answer 27

shine light at one wavelength at the cells and it reflects it at different wavelength filters are used too

Question 28

how are antibbodies ued in fluorescent microscopes?

Answer 28

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

Question 29

how is the location of the fluorescence detected in the tissue?

Answer 29

microscopes

Question 30

why is live cell imaging used?

Answer 30

cells are dynamic and processes occur over time | want to visualise these processes rather than a snapshot of them

Question 31

how does live cell imaging work?

Answer 31

uses an inverted microscope | objective is placed under the micscope allowing view of cells in culture media

Question 32

how are cells kept alive in live cell images?

Answer 32

environment chamber maintains the temp, adjusts gas suply to maintain pH kept alive and health for several days under the microscope

Question 33

what is GFP

Answer 33

a gene from a jellyfish

Question 34

what was the original form of GFP like?

Answer 34

worked best at sea temps and onyl weakly fluoreced, easyily bleached and unstable

Question 35

what is gfp like now?

Answer 35

mutant version workes at 37degrees and can now work as a monomer and is more photostable

Question 36

what are the uses of gfp?

Answer 36

track embryo development | protein dynamics

Question 37

what develoments are there for gfp now?

Answer 37

multi colours of it now can be used to track multiple things eg brain neurones all together

Question 38

what is FRAP

Answer 38

fluorescence recovery after photobleaching

Question 39

when is FRAP used?

Answer 39

determine kinetics of protein redistrubution and detect how much of the proteinis immobile or membrane bound

Question 40

what other types of gfp are there?

Answer 40

RFP | isolated from coral

Question 41

what is intravital imagining?

Answer 41

the use of GFP in cells or tissues in live organisms being viewed

Question 42

what is photo activation

Answer 42

laser shined onto tissues and changed colour after stimulation allows tracking of sub-population cells

Question 43

what arethe main limitations of photoactivation?

Answer 43

- photobleaching - phototoxicity - overexpression - protein folding - cellular distrubution

Question 44

what is photobleaching?

Answer 44

limitation of photoactivation | fluorescent proteins are sensitive to bleaching so long term studies can be tricky

Question 45

what is photo-toxicity

Answer 45

limitation of photoactivation | illumination of cells with high-intensity light can be toxic formin free radicals

Question 46

what is overexpression?

Answer 46

limitation of photoactivation tagged genes are often expressed at high levels to make a good signal excess proteins can after the cells normal distrubution

Question 47

what is cellular distribution in relation to photoactivation?

Answer 47

the large fluorescent proteins may affect the cellular disrubution of the tagged proteins which is especially key for membrane proteins

Question 48

what is a confocal microscope?

Answer 48

modified version of fluorescent miscopscopy

Question 49

how does a confocal microscope work?

Answer 49

lasers of specific wavelength are used to illuminate molecules in the sample 3D image of the cell with cross sections

Question 50

advantages of a confocal microscope

Answer 50

- 3D view - cross sections - info on spatial relationship - used on both cells and tissues

Question 51

advantages of TEM

Answer 51

highest resolution possible

Question 52

how does TEM work?

Answer 52

antibodies are labelled with gold to detect cellular structures

Question 53

the disadvantage of TEM

Answer 53

requires very thin sections of cells

Question 54

what is super resolution microscopy?

Answer 54

provides 3D view of cells within a tissue and looks at spatial relationships within the tissue

Question 55

can super resolution or confocal do timelapses?

Answer 55

no | laser light is scattered by skin and other tissues and does not penetrate much

Question 56

what is STORM?

Answer 56

stochastic optical resolution

Question 57

what does STORM do?

Answer 57

allows you to determines the exact centre of the fluorescent signal and therefore the exact location o the dye molecule

Question 58

limitations to STORM

Answer 58

can be very slow photobleaching and toxicity can be an issue limited use of GFP expensive