Lecture 2: Introduction to Cell Culture and Proteins

Question 1

Cell culture refers to the…

Answer 1

removal of cells from an animal or plant & the process by which these are grown in a favourable artificial environment-controlled conditions, generally outside of their natural environment

Question 2

The conditions vary but the environment that is made to control these conditions consist of a…

Answer 2

suitable vessel like a flask, dish or well that contains the substrate, the medium that supplies the essential nutrients, growth factors and hormones and an incubator that controls humidity, CO2 and temperature.

Question 3

Sterile tissue culture techniques are crucial to…

Answer 3

preventing contamination

Question 4

The non-adherent cells are…

Answer 4

suspended in liquid as single cells or free floating clumps with the advantage of this being a large number of them can be cultured in a very small space

Question 5

Adherent cells grow in a…

Answer 5

monolayer meaning cell growth is limited by the surface area. The cells are released from the dish by enzymatically or mechanically detaching them from the dish.

Question 6

A good indicator of how well cells are growing

Answer 6

Density

If left unattended, the increased density can cause overcrowding and the flask must be passaged (sub-culturing cells to dilute them by lifting them, diluting the volume and removing some of the cells). This is because the cells become stressed and eventually detach and die. A healthy environment for cell growth is for them to always been in the log-phase of growth.

Question 7

Confluency

Answer 7

A measure of the percentage of the surface area covered in cells

Question 8

Passage number

Answer 8

The number of times a cell culture has been sub-cultured. Some cell lines can only be grown for a finite number of passages.

Question 9

Cell release describes the way cells may be

Answer 9

detached or released from the flask/dish either mechanically or enzymatically

With the enzyme technique, trypsin is used to cleave peptide bonds in fibronectin of the extracellular matrix. EDTA is used to chelate calcium ions in the media that would normally inhibit trypsin.

Mechanical detachment involves the use of a cell scraper.

Question 10

However, trypsinizing too long will reduce

Answer 10

cell viability

Question 11

Cell detachment is carried out in order to…

Answer 11

Quantify cells

This is done by counting a sample that has been detached in a haemocytometer using a counter to determine cell density.

Question 12

Cell seeding

Answer 12

When the number of cells has been determined, cells can be seeded for an experiment.

It is to spread cells to a culture vessel to enable the performing of experiments.

Question 13

The proteins within cells can be isolated from the cells grown by protein purification

Answer 13

The cells are first treated with a particular variable including drugs, DNA damaging chemicals and chemotherapy before the cell is lysed by detergents and sonification to expose the proteins.

The proteins can then be isolated from different extracts including whole cell, nuclear and cytoplasmic extracts.

The latter 2 extracts are derived by using different strengths of detergent to isolate the proteins of interest.

Question 14

Harvesting cells describes the…

Answer 14

Mechanical lifting of cells using a cell scraper

The cells are removed from media, are then scraped into PBS and then centrifuged to get a cell pellet. The reason this is sometimes preferred over trypsin is that trypsin itself can alter the phosphorylation state of some of the proteins found on the cell surface so if these proteins are being researched, trypsin cannot be used and this mechanical technique is used instead

Question 15

HeLa cells became one of the first…

Answer 15

immortal cell lines and contaminated most cell lines of the time due to poor technique (1951)

They did however lead to the discovery of many different cancers, viruses, facilitated advances in in vitro fertilization, cloning and in gene mapping.

Question 16

Central Dogma

Answer 16

The flow of genetic information from DNA to the cytoplasm

Question 17

In eukaryotes, the processes of transcription and translation are compartmentalised due to the presence of the…

Answer 17

A nuclear membrane

Question 18

Proteins are involved in most enzymatic reactions within the body

Answer 18

Proteins synthesis is a very expensive process for the cell and is therefore tightly regulated. This regulation can control the overall rates of proteins synthesis as well as modulate the initial expression of the genes at the transcript level (e.g. regulation from the promoter).

Protein synthesis is inhibited by cell stresses and the withdrawal of nutrients. This is what is utilised to regulate cell growth in culture.

Question 19

Cell stress can be induced by a number of techniques including

Answer 19

…serum deprivation, temperature shock, DNA damage, viral infection, hypoxia and cytokine treatment. With enough stress, apoptosis can be induced.

Question 20

The 80S ribosome is a combination of…

Answer 20

…the small and large ribosomal subunits that bind together during initiation of translation

Question 21

Translation itself takes place in the…

Answer 21

cavity between the two subunits

Question 22

80S has

Answer 22

3 binding sites for tRNA

The small and large subunits continually associate and dissociate.

Question 23

What is on the 60S (large ribosome)?

Answer 23

Peptidyl transferase on the 60S (large) subunit associated with the regulation of translation as it links amino acids together during elongation

The small and large subunits continually associate and dissociate.

Question 24

A polysome is a structure that consists of

Answer 24

multiple ribosomes attached to a single mRNA with up to 10 ribosomal units able to bind to one transcript

Question 25

Proteins can be obtained from a range of sources including

Answer 25

patient cells or tissues, microorganisms a cell lines that are grown in culture.

Question 26

Cells are lysed to...

Answer 26

...isolate proteins of interest and depending on the strength of the detergent, different extracts can be obtained If SDS is used for example, both the cell and nuclear membranes are lysed meaning the sample obtained is a whole cell extract. Using more gentle detergents like triton, only a cytoplasmic extract is obtained.

Question 27

Protein extracts are quantified through...

Answer 27

Bradford assays Bradford reagent is red in the absence of protein and is blue if protein is present. A standard for different concentrations is then produced using known concentrations of protein with this then graphed. The unknown quantity of proteins in different samples is then determined using this standard (colorimetric assay).

Question 28

Bradford reagent is ___ in the absence of protein and is ___ if protein is present.

Answer 28

Bradford reagent is red in the absence of protein and is blue if protein is present.

Question 29

Once the protein has been quantitated, the proteins within the extract are separated via...

Answer 29

SDS-polyacrylamide gel electrophoresis This is a discontinuous gel system with the stacking gel having a different pH to the separating gel (slightly higher). The reason for this is glycine that is the solution the proteins are in causes the cells to stack at a lower pH but then separate out at a higher pH. The sample buffer in which the protein is put in before electrophoresis is at the pH of the stacking buffer, have the equal volumes, has bromophenol blue to visualise and glycerol to give the proteins some density. There is also SDS that is an anionic detergent (breaks non-covalent bonds) and adds a negative charge to the amino acid side chain to facilitate the migration of proteins to the anode within the gel. Beta-mercaptoethanol is also used and this breaks down disulphide bonds within proteins to allow them to migrate.

Question 30

The gels from electrophoresis can either be...

Answer 30

...stained to visualise all the proteins within the sample or undergo semi dry blotting to allow the transfer of proteins from the gel to a membrane. The gel is put on a membrane and this is sandwiched with filter paper and then a cathode on one side and an anode on the other. When current is applied, the protein will then migrate to the membrane and be fixed on to it.

Question 31

The protein fixed to the membrane can then undergo **western blotting** - what happens in this technique ?

Answer 31

A primary antibody is introduced to the membrane to bind to the protein of interest. This is then visualised through the introduction of a conjugated secondary antibody that allows for visualisation after the introduction of a substrate. This can then be captured using X-ray film.

Question 32

The general difference between staining and a western blot is that with \_\_\_\_\_\_?

Answer 32

Staining All proteins within the extract are visualised and this can determine if the samples are equally loaded. It cannot however identify specific proteins. A Western blot can identify specific proteins and can also be re-probed to look for other proteins. This process is also semi-quantitative through the use of software to quantitate bands.

Question 33

COSHH regulations 2002

Answer 33

For safety standards within the lab (see slides for details) ## Footnote COSHH is the law that requires employers to control substances that are hazardous to health. You can prevent or reduce workers exposure to hazardous substances....