Lecture 5- How are cells studied?

Question 1

How are unicellular eukaryotes studied? i.e S. cerevisiae

Answer 1

Cultures are grown in YEPD liquid media or on YEPD agar plates

Question 2

YEPD media

Answer 2

Yeast
Extract (ground up yeast cells)
Peptone (milk proteins)
Dextrose (glucose)

Question 3

Most animal and plant cells can live, grow, and multiply in vitro if provided with appropriate media

True or False

Answer 3

True

Question 4

What are needed to grow cells in culture?

Answer 4

amino acids

vitamins

salts

glucose- carbon source, ATP

antibiotics- kill bacteria

growth factor proteins- cell reproduction

Question 5

BPAE cell line

Answer 5

made in 1994 from a cow heart

Question 6

HeLa cell line

Answer 6

used to study multicellular eukaryotes

Question 7

Subcellular fractionation

Answer 7

the isolation and purification of cellular organelles and macromolecules using centrifugation

Question 8

3 steps of subcellular fractionation

Answer 8

1. Homogenization
2. Isolation
3. Purification

Question 9

Homogenization

Answer 9

breaks the plasma membrane to release the cell contents

whole cells -> cell homogenate

when carefully applied, homogenization leaves most of the membrane-enclosed organelles intact

Question 10

4 methods of homogenization

Answer 10

cell suspension or tissue ->

1. break cells with high-frequency sound (sonication)
2. use a mild detergent to make holes in the plasma membrane
3. force cells through a small hole using high pressure
4. shear cells between a close-fitting rotating plunger and the thick walls of a glass vessel

Question 11

Isolation

Answer 11

separates cell components according to whether they sediment or not

cell homogenate (before centrifugation) -> supernatant (top) and pellet (bottom)

supernatant: smaller and less dense components
pellet: larger and more dense components

Question 12

Up to 4 centrifugations may be required to isolate the desired organelles/ macromolecules

Answer 12

cell homogenate- use low-speed centrifugation to obtain pellet 1

pellet 1: whole cells, nuclei, cytoskeletons

supernatant 1 - use medium-speed centrifugation to obtain pellet 2

pellet 2: mitochondria, lysosomes, peroxisomes

supernatant 2 - use high-speed centrifugation to obtain pellet 3

pellet 3: microsomes, other small vesicles, ER

supernatant 3- use very high-speed centrifugation to obtain pellet 4

pellet 4: ribosomes, viruses, large macromolecules

Question 13

Microsomes

Answer 13

pieces or fragments of ER

Question 14

Purification- Velocity centrifugation

Answer 14

purifies cell components according to speed

sample is at the top

fast-sedimenting component appears at the bottom

slow-sedimenting component appears at the top

centrifuge tube pierced at its base

automated rack of small collecting tubes allows fractions to be collected

Question 15

Purification- Equilibrium centrifugation

Answer 15

purifies cell components according to buoyant density

the sample is distributed throughout the sucrose density gradient

at equilibrium, components have migrated to a region in the gradient that matches their own density

Question 16

How many ways can you obtain mitochondria?

A. 1
B. 2
C. 4
D. 8
E. 16

Answer 16

D. 8

4 methods of homogenization including sonication
1 method of isolation (differential centrifugation)
2 methods of purification (velocity centrifugation or equilibrium centrifugation)