Assays to Quantitate Bone Differentiationweek 13

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Assays to Quantitate Bone Differentiationweek 13
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Biojewelry update

• http//www.biojewelry.co.uk/

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Spectrophotometry

• Transmitted light passed through a solute
• Can measure light that has been transmitted or
  absorbed (by the sample)
• Darker reactions will absorb more light
• Ax will be higher
• A blank is used to eliminate any absorbance
  caused by solvent or buffer
• Helps determine if there is a contamination or
  cross-reaction in your buffer
• In our case, PBS used to create lysate

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Repeat samples

• Statistically, error is possible
• Sources of error include
• Pipetting errors
• Mixing errors
• Faults in the plate
• Plate reader (?)

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Nomenclature

• Absorbance is often reported as Al.
• Transmittance is reported as T
• Optical Density (OD) is the relationship between
  transmittance and the concentration of the
  colored compound
• We will only be using Absorbance
• Compounds created by sample and reagent have
  optimal wavelengths where absorbance occurs.
• ALP is read at 405nm
• Calcium is read at 630nm

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More Nomenclature

• When you use a well of everything in your sample
  solution MINUS the actual sample this is referred
  to as
• Blank
• or Zero
• or Reference
• Standards are solutions of known concentration
  used to calibrate the readings

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ALPalkaline phosphatase

• p-Nitrophenyl Phosphate is the reagent used to
  indicate concentration of alkaline phosphatase in
  the sample
• ALP supposed to peak at 2 week and diminish as
  mineral is laid down
• This assay will determine if this is true or not

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ALP formula

• Unit of activity (U/L) -That amount of enzyme
  causing the hydrolysis of one micromole of
  p-nitrophenyl phosphate per minute
• ALP(U/L) DA/minute X TV X 1000
• 12.72 X SV
• TV total volume (0.270ml)
• 1000 conversion of units from ml to L
• 12.72 millimolar absorptivity of p-nitrophenol _at_
  405nm _at_ 25oC
• SV sample volume (variable)
• Millimolar absorptivity is known so a standard is
  not required
• PBS sample acts as a zero
• Not included in calculation

ALP H2O ? Na2HPO4
H
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Notes for ALP assay

• This sample is retrieved from entire lysate
• Note that the sample volumes vary for the
  samples. This changes step 2 on page 110 of the
  manual.
• This was an adjustment made to keep the readings
  within the parameters of the plate reader.
• 1 week and undifferentiated 0.01ml
• 2 week and 3 week 0.005ml

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Other notes

• Keep your aliquoted lysates on ice block
• TA will add RT reagent (p-nitrophenyl phosphate)
• 1 group/plate
• Vortex (mixed) lysate sample
• Timed assay, 0, 2, 4, 6, 8 and 10 minutes
• Reaction develops over time

• Also required is Standard Deviation calculation
  for each average
• Not included in the lab manual at this time
• X sample absorbance
• X average absorbance
• n number of readings

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Calcium detection

• Optimal l is 612nm but reading within a range is
  acceptable
• Requires creation of a linear standard curve
• Absorptivity of reaction reagent is unknown
• TA will run standard sample of different
  concentrations for each plate
• Must run standards each time you run a plate.

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Calcium assay formula

• Ca2 of sample (mg/dL)
• Asample Ablank
• slope
• Blank is in well H 1 of each plate
• There is calcium reagent, but no calcium standard
• Linear detection range 0.08 mg/dL (0.02mM) to 20
  mg/dL (5mM) calcium in 96-well plate assay.

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?QuantiChrom Calcium Assay Kit (BioAssay Systems)
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Other notes for Calcium Assay

• 3 groups/plate since reaction is stable for 60
  minutes
• Centrifuged supernatant is required sample
• TA will load standards and all calcium reagent
• Calcium content should increase over time
  (undifflt1wklt2wklt3wk)

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Cell Migration
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Cell Migration

• Important in physiological and pathological
  processes
• Organogenesis and embryonic development
• Wound healing and angiogenesis
• Immune system
• Cancer metastasis
• Chemotaxis
• Cell movement in a soluble concentration gradient

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Tissue Engineering Palsson, Bhatia 2004
These five steps occur after cell is polarized
toward chemoattractant
Thin membrane filaments that are pulled out of
the cell as it moves away are called Retraction
fibers
PtK1 cell
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ECM

• Cell interactions with ECM exert control over the
  function of the adherent cells
• Cell spreading, adhesion and migration
• Surface density of adhesion proteins influences
  the rate of cell migration
• Too low intracellular contraction forces
  dominate and prevent effective traction against
  the substrate.
• Too high adhesion strength excessive for cell
  migration, the rear of cell is not released and
  cell speed is reduced

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Tissue Engineering, Minuth, Strehl, Schumacher
2005
Principles of Tissue Engineering, Lanza, Langer,
Vacanti 2000
Tissue Engineering, Minuth, Strehl, Schumacher
2005
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Migrating cell images from 2006
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Measuring cell motility

• Time lapse microscopy
• Tracking individuals and populations at fixed
  intervals
• Specialized migration chambers
• Before and after measurements by placing cells in
  a starting chamber and counting the cells that
  crawl to another location in a defined period
• Micropatterning techniques

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Migration, Boyden Assay

• Cells above the membrane, chemoattractant is
  below ? concentration gradient
• Pores 3-8 microns (large enough for the cells to
  squeeze through, small enough so they do not
  fall)
• Give time to migrate, fix and stain the membrane,
  and count cells at the bottom
• Disadv cannot trace migration
• Adv good to view population migration

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Migration, Zigmond chamber
one well has chemoattractant
cells are under the coverslip, observe them from
the top while migrating
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Migration, Dunn

• similar to Zigmond in principle, wells are
  concentric as opposed to linear

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Migration, Under agarose
Chemoattractant diffuses through the agarose and
cells migrate out of their well under the agarose
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Cell Migration in Tissue Engineering

• Polymeric scaffolds created and implanted
• tissue engineers hope to select certain cell
  types over others
• Induce enhanced migratory responses of particular
  cell types by
• Surface chemistry
• Release of chemoattractants from within the
  implanted material