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Magnetic Twisting Cytometry: Cell Mechanics Evaluation Technique

This approach applies torque to cells using magbeads to assess cellular viscoelasticity and mechanotransduction. The apparatus involves a microscope stage with electromagnetic coils and human airway smooth muscle cells with magbeads. A coercive field is briefly applied in one direction, while a sinusoidal field is applied in a perpendicular direction to twist the cells. The torque response is measured using a magnetometer. Histamine and DibutryrlcAMP are used to evaluate the stiffening and softening of cells. The technique allows for tracking individual particles and assessing the firm integrin and CSK connection. Firm CSK inhibition is necessary for free twisting. Rheological measurements include complex modulus and storage and loss moduli calculations. Wide variability in cell response highlights the need for large sample populations. Cells with different baseline characteristics show varying responses to stiffening and softening stimuli.

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Magnetic Twisting Cytometry: Cell Mechanics Evaluation Technique

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  1. Session 10 Magnetic twisting cytometry

  2. Approach • Torque applied at surface of cells with magbeads • Used to determine cell mechanics • Cellular viscoelasticity (Fabry, Fredberg) • Mechanotransduction (Wang, Ingber)

  3. Technique • Coercive field applied briefly in one direction • Sinusoidal field applied in perpendicular direction • Torque applied: t = m x H M is magnetic moment H is sinusoidal external field

  4. Apparatus A) Microscope stage with electromagnetic coils B) Twist causes magbead rotation and translation C) Human airway smooth muscle cells with magbeads

  5. Magnetic Measurement • Magnetometer detects remanent magnetic field, B • Twisting field rotates magbeads • Magbead’s m turned away from magnetometer • Decrease in B field from horizontal direction indicates degree of twist

  6. Torque Response • For magbeads: T(t) = c H(t) cos (t) c = 0.41 Pa/gauss H(t) = H sin (t) (t) = B(t)/B0 Histamine added First 10 cycles Pre-histamine Last 20 cycles Post-histamine

  7. Particle Tracking Measurement • Fast, real-time image analysis • Individual particle tracking better than aggregated magnetometry • Pixel-to-displacement, intensity weighted algorithm of centroid position per frame

  8. Firm Integrin and CSK connection • Ligand-bound integrins actively resist the twist

  9. Full CSK Needed for Twist Resist • CSK Inhibitors • Acrylamide (Acr) inhibits IF • Nocodozal (Noc) inhibits MT • Cytochalasin D (Cyt) inhibits actin • Full inhibition of CSK needed for “free” twisting

  10. Rheological Measurements • Complex Modulus: (s, Fourier transform) • Magnetometry: G*(s) = T(s) / (s) • Optical Tracking: G*(s) = T(s) / x(s) • Storage and Loss Moduli: G*(s) = G’(S) + iG’’(s) • Hysteresivity : h (s) = G’’(S) /G’(s)

  11. Histamine Stiffens hASMCs • Histamine activates myosin, leads to SM contraction • Steady baseline before adding histamine • Storage modulus increased 2.2X • Loss modulus increased 3X • Transient hysteresistivity response

  12. DibutryrlcAMP softens hASMCs • DBcAMP inhibits myosin, leads to SM relaxation • G’ falls to 30% • G’’ decreased to 45% • H increased 1.5X

  13. Wide Variability in Cells • Not donor-to-donor, day-to-day, or culture-to-culture, but cell-to-cell variability • Wide distribution in data necessitates large sample populations DBcAMP-softening Histamine-stiffening

  14. Baseline Affects Stiffening/Softening • “Soft” baseline cells: More histamine-stiffening, less DBcAMP-softening • “Hard” baseline cells: More DBcAMP-softening, less histamine-stiffening

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