Cell culture and an Overview of techniques used to isolate proteins

Question 1

What is cell culture?

Answer 1

Cell culture refers to the removal of cells from an animal or plant and the process by these are grown in a favourable artificial environment- controlled conditions, generally outside their natural environment

Question 2

Culture conditions vary widely for each cell type, but the artificial environment
in which the cells are cultured invariably consists of?

Answer 2

•Suitable vessel (flask or dish or well)
containing a substrate or
• Medium that supplies the essential nutrients
(amino acids, carbohydrates, vitamins, minerals),
growth factors, hormones etc.
• Incubator (humid CO2 incubator at the
appropriate temperature)

Question 3

What is the difference between adherent and non adherent cells in vitro?

Answer 3

Non - adherent:
Suspension cells are suspended in liquid as single cells or free floating clumps.

Adherent:
Adherent cells grow in a single layer called a monolayer.
Cell growth is limited by the surface area.
Cells may be released from the dish enzymatically or mechanically.

Question 4

What are the 4 methods of maintaining cells in culture?

Answer 4

• Density
Cells are grown and maintained using the appropriate
conditions. When they get too dense for the flask must be passaged (sub-cultured)

• Confluency
Cell density can be a measure of proliferation. It is usually
combined with an estimated (or counted) percentage, so 10% confluency means that 10% of the surface the dish or flask used is covered with cells, 100% means that it is entirely
covered.

• Cell passage (sub-culture)
Involves lifting the cells, diluting the volume and removing some of the cells.

• Passage number
is 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 5

What 2 ways can cells be released? Explain each.

Answer 5

Cells may be released or detached from the flask/dish enzymatically (trypsin EDTA) or mechanically.

• Enzymatically
Trypsin cleaves peptide bonds in fibronectin of the extracellular matrix
EDTA chelates calcium ions in the media that would normally inhibit trypsin
Trypsinizing too long will reduce cell viability.

• Mechanically
Cell scraper

Question 6

How can cells be quantified?

Answer 6

Cells can be quantified by counting a sample in a haemocytometer using a counter to determine cell density

Question 7

What are the different extracts that a protein can be purified and analysed from?

Answer 7

• Whole cell
• Cytoplasmic
• Nuclear

Question 8

Briefly explain how to harvest cells

Answer 8

Mechanically lifting cells- using cell scraper
Remove media from cells
Harvest cells by scraping into PBS
Centrifuge to get a cell pellet

Question 9

What is the central dogma?

Answer 9

The Flow of Genetic Information from Nucleus to Cytoplasm.
AKA DNA > RNA > Protein
1) Synthesis of mRNA in the nucleus
2) Movement of mRNA into cytoplasm via nuclear pore
3)Synthesis of protein

Question 10

Why is protein synthesis so important?

Answer 10

Crucial for cell growth, proliferation and survival
Expensive process for the cell, therefore tightly
regulated. (Energetically expensive)
Regulation can control overall rates of protein
synthesis and modulate the expression of specific
transcripts.
Inhibited by cell stresses and the withdrawal of
nutrients:-serum deprivation, temperature shock,
DNA damage, viral infection, hypoxia, cytokine
treatment.

Question 11

what is a polysome?

Answer 11

a structure that consists of multiple ribosomes attached to a single mRNA

Question 12

What are some different sources of protein?

Answer 12

Proteins can be obtained from a wide variety of samples. For diagnostic purposes, they may be obtained from:
• Patient cells or tissues
• Microorganisms
• Cell lines derived from insects, vertebrate animals, or plants
- cell culture

Question 13

What are some examples of detergents used in cell lysis?

Answer 13

Triton X100, NP40, Tween 20 and SDS

Question 14

briefly describe lysis of cell pellets

Answer 14

pellet cultured cells
incubate
centrifuge
collect supernatant (protein extract)

Question 15

what are the components of a sample buffer ( SDS page)

Answer 15

SDS - Anioinic detergent, when it dissolved gives a net negative charge. Functions: breaks non covalent bonds, binds to amino acid side chains giving a net negative charge

B-mercapthoethanol - reducing agent. Reduces disulfide bonds in proteins

Bromophenol blue
Glycerol
ddH20
Tris-HCL pH 6.8

Question 16

What can we identify with stained gels vs blots?

Answer 16

Stained gel:
Can stain all protein within extract
Can also tell you if samples are equally loaded
Cannot identify specific proteins

Blots:
Can identify specific proteins with an extract
Can also probe to look for loading
Semi-quantitative, can use software to quantitate bands