PT2: 28/09

Question 1

What is an advantage of applying osmotic stress to cells? What is an assumption?

Answer 1

Advantage: stress is applied uniformly across the cell.
Assumption: Osmotic pressure gradient spatially constant

Question 2

What is the most popular solute for controlled osmotic stress?

Answer 2

PEG

Question 3

What was the overall aim of their project?

Answer 3

To use AFM to measure cell volume change in response to osmotic stress to quantify mechanical cell modulations

Question 4

What is the general protocol timeline of this project?

Answer 4

1. Preparation of polyacrylamide (PA) substrates
2. Cell culture and plating for AFM
3. Osmotic pressure induction
4. AFM volume measurement
5. Translation to mechanical properties

Question 5

What are the favorable features of PA?

Answer 5

• Deforming in proportion to the applied stresses
• Recovering completely on release of force
• Allowing systemic and reproducible control of flexibility of substrate
• Using specific ECM molecules as ligands for cell adhesion, while PA does not interact with the cell surface
• Porous which allows penetration of media

Question 6

What are disadvantages of PA?

Answer 6

Adheres poorly to glass, so it must be covalently attached to the glass surface by chemical activation of the glass surface

Question 7

How is the activated glass surface prepared?

Answer 7

need to add

Question 8

How is osmotic pressure induced in the system?

Answer 8

Hyperosmotic stress is applied by adding PEG 400 to the isotonic culture medium

Question 9

What are the two AFM methods to measure volume?

Answer 9

1. Geometric: measure dimensions from height profiles and then apply geometric models
2. Direct: measure volumes directly from images, but background correction is required

Question 10

What equation relates osmotic stress and cell volume?

Answer 10

Boyle-Van’t Hoff Equation
- add equation to study as well

Question 11

What is the equation which relates shear modulus to bulk modulus?

Answer 11

G is related to B by the poisson ratio (v)

G = 3/2 [B(1-2v)/(1+v)]

Question 12

What cell stiffening results did they see?

Answer 12

In in G vs. time graphs, there is first an exponential increase, then a plateau is established once Vmin is reached, and it recovers upon removal of osmotic stress

Question 13

What are the benefits of using osmotic stress for cell mechanics measurements?

Answer 13

• Uniform, tunable osmotic stress
• Analogous to contractile compression
• Reproducible with different volume measurement methods
• Material and cost advantage

Question 14

What are the limitations of using osmotic stress for cell mechanics measurements?

Answer 14

• Underestimation of absolute height by AFM
• Water efflux varies by cell
• Crosslinked cytoskeleton stores much larger elastic energy than that in the cortex