PT2: 28/09 Flashcards
What is an advantage of applying osmotic stress to cells? What is an assumption?
Advantage: stress is applied uniformly across the cell.
Assumption: Osmotic pressure gradient spatially constant
What is the most popular solute for controlled osmotic stress?
PEG
What was the overall aim of their project?
To use AFM to measure cell volume change in response to osmotic stress to quantify mechanical cell modulations
What is the general protocol timeline of this project?
- Preparation of polyacrylamide (PA) substrates
- Cell culture and plating for AFM
- Osmotic pressure induction
- AFM volume measurement
- Translation to mechanical properties
What are the favorable features of PA?
- 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
What are disadvantages of PA?
Adheres poorly to glass, so it must be covalently attached to the glass surface by chemical activation of the glass surface
How is the activated glass surface prepared?
need to add
How is osmotic pressure induced in the system?
Hyperosmotic stress is applied by adding PEG 400 to the isotonic culture medium
What are the two AFM methods to measure volume?
- Geometric: measure dimensions from height profiles and then apply geometric models
- Direct: measure volumes directly from images, but background correction is required
What equation relates osmotic stress and cell volume?
Boyle-Van’t Hoff Equation
- add equation to study as well
What is the equation which relates shear modulus to bulk modulus?
G is related to B by the poisson ratio (v)
G = 3/2 [B(1-2v)/(1+v)]
What cell stiffening results did they see?
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
What are the benefits of using osmotic stress for cell mechanics measurements?
- Uniform, tunable osmotic stress
- Analogous to contractile compression
- Reproducible with different volume measurement methods
- Material and cost advantage
What are the limitations of using osmotic stress for cell mechanics measurements?
- Underestimation of absolute height by AFM
- Water efflux varies by cell
- Crosslinked cytoskeleton stores much larger elastic energy than that in the cortex
