Culturing (Lecture 4)

Question 1

Why is solid surface good for culturing animal cells

Answer 1

CAMs are given a solid surface to grow

Question 2

What type of media provides nine essential amino acids and often has bacteria added to reduce contamination?

Answer 2

Solid surface

Question 3

WHat type of media includes inorganic salts, a carbon source water and tends to be faster growing for cells?

Answer 3

Suspension

Question 4

What type of culture environment needs control temperature, atmosphere humidity and to keep strict sterile conditions?

Answer 4

Solid Surface

Question 5

What type of culture media needs a control temperature, can use standard laboratory conditions and be kept moderately sterile?

Answer 5

Ssupension

Question 6

______ cells are isolated directly from tissues (skin, kidney or liver)

Answer 6

Primary

Question 7

True or False: Primary cells have a finite life span,

Answer 7

True

Question 8

What are the three phases of human cell growth (not embryonic stem cells)?

Answer 8

Phase I (initial growth)
Phase II (Cell strain)
Phase III (Cell Senescence)

Question 9

What are the three phases of mouse cell growth?

Answer 9

Initial loss of growth potential
Senescence
Emergence of immortal variant (cell line)

Question 10

______ is an abnormal number of chromosomes.

Answer 10

Anueploidy

Question 11

What are the three steps to isolating organelles?

Answer 11

1. Lyse the cells
2. Separate organelles based on key characteristics
3. Use isolated organelles for further study

Question 12

What are 3 ways to lyse a cell?

Answer 12

High-speed blending
—Sonication
Tissue homogenizer

Question 13

True or False: Swelling cells in a hypotonic solution weakens the plasma membrane, making it easier to rupture.

Answer 13

True

Question 14

______ allows molecules to freely across the plasma membrane, along their concentration gradient.

Answer 14

Simple diffusion

Question 15

______ (channels, carriers) allows molecules to move through a membrane protein, along their concentration gradient.

Answer 15

Facilitated diffusion

Question 16

After lysis, mix of suspended cellular components is the ______.

Answer 16

Homogenate

Question 17

True or False: The homogenate can be kept at room temperature.

Answer 17

False, kept a cool temperature so enzymes are not denatured by proteases

Question 18

______ techniques separate particles based on mass or density.

Answer 18

Centrifugation

Question 19

______ of a cell homogenate yields fractions of organelles that differ in mass and density.

Answer 19

Sequential differential centrifugation

Question 20

______ separates cellular components by density.

Answer 20

Equilibrium density-gradient centrifugation

Question 21

True or False: Density-gradient centrifugation is the most common initial step in protein or organelle purification.

Answer 21

False, differential centrifugation

Question 22

How can you purify a sample of organelles?

Answer 22

Antibody/organelle complex can be coated on metallic beads, and then pelleted with a magnet

Antibody/organelle complex can be absorbed to killed bacteria and then pelleted by centrifugation

Question 23

True or False: Organelle specific antibodies can recognize soluble proteins inside the vesicle.

Answer 23

False

Question 24

What are the three steps of proteomics analysis of organelles?

Answer 24

1. Isolation of organelle at high purity
2. Method to identify all proteins in the organelle
   Digest with protease-like trypsin (cuts at Lys & Arg)
   — Determine mass and sequence of peptides using mass
   spectrometry to generate a ‘fingerprint’
3. Genome sequence for reference comparison
   — Provides ‘list’ of all possible proteins
   — Mitochondria, chloroplast etc have their own separate genome that would need to be referenced

Question 25

How can you tell which molecule is mitochondria based on proteomics.

Answer 25

Organelles have recognizable structure and contain characteristic proteins to perform its function

Therefore there would be a presence of ATP synthase

Question 26

True or False: The best markers are the ones found in the organelle of interest and not found in any other organelle compartments.

Answer 26

True