BIOE 202 Section AB1

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BIOE 202Section AB1

• Lab 13
• ALP and Calcium AssaysCell Migration

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Lab 13 Overview

• Review protocols and information
• Alkaline Phosphatase and Calcium Assays
• Cell Migration

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Lab Reports

• Last lab report due Thursday, May 3rd by 5pm
• Only one submission is allowed
• Will cover differentiation of mesenchymal stem
  cells to bone and analysis of mature bone
  characteristics (labs 9-13)
• Note hair follicles and neuron labs are not
  included

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A1. ALP AssayLab manual p. 115

• Load lysate (5 or 10 µL) work quickly
• Add ALP reagent
• Take absorbance readings at 405nm every 2 minutes
  for 10 minutes
• Record and graph readings

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A2. Calcium AssayLab manual p. 119

• Load lysate supernatant
• Add calcium reagent
• Take absorbance readings at 630nm
• Data should be graphed and pasted into your
  notebook

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B. Cell MigrationLab manual p. 123

• Assemble Zigmond chamber
• Load 3T3-L1 cells and chemoattractant
• Incubate in 5 CO2
• Record observations and take a photo

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Return Day

• No Return Day!

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Reminders

• Final exam during class April 30th
• Hand written note sheet one side of 8.5x11
• Third lab report due May 3rd at 5pm

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• Thanks for a great semester!

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Order of Events

• All groups begin Alkaline Phosphatase assay
• Calcium Assay
• Cell Migration

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Online Quiz Results

• In spectrophotometry, the well that contains
  everything except the sample is called the blank
• The solution of known concentration is called the
  standard
• The absorbance for the alkaline phosphatase
  sample reactions and microplate reading is read
  at a wavelength of 405nm
• The blank well for the calcium reaction contains
  a known volume of calcium reagent but no calcium
  standard
• The calcium sample from osteoblast lysates is
  retrieved from supernatant of a centrifuged
  sample and needs to be kept on ice
• The Boyden assay is most beneficial to study cell
  population migration through a filter with holes
  3-8 microns in size
• The effect of too high of an adhesive substrate
  density on cell migration is excessive adhesion
  strength for cell migration because the rear of
  the cell isnt released so cell speed is reduced
• Chemotaxis is used to describe cell movement in a
  soluble concentration gradient
• The five steps of cell migration are extension,
  attachment, contraction force, release, and
  recycling