BIOE 202 Section AB1

1
BIOE 202 Section AB1

• Lab 12 Staining of Constituents of Bone Created
  by Differentiated Osteoblasts and Formation of
  Bone Lysate
• April 14th, 2008

2
Lab 12 Overview

• Lab 12 Online Quiz Results
• Review Lab 12 Protocol
• Final Reminders and Dates
• Video Miracle Cell
• Complete Lab Exercises
• Clean Up!

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Online Quiz Results

• Osteoblasts secrete alkaline phosphatase to
  increase the local concentration of phosphate
  ions
• Osteoblasts secrete osteocalcin to increase the
  local concentration of calcium ions
• Collagen is the main component of osteoid
• In vivo most unmineralized bone osteoid is found
  just underneath the periosteum
• Every 3 days new osteoblasts push older
  osteoblasts toward mineralized bone and encase
  them
• The D1 cells are performing intramembranous
  ossification when differentiating
• In vivo bone formation absolutely requires
  vascularization to have a source of calcium ions,
  but we are forming bone in vitro, which does not
  require vascularization
• Von Kossa staining involves fixing with NFB,
  staining for ALP with red violet, and staining
  for mineralized bone with silver nitrate
• The pipet for silver nitrate requires special
  disposal

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Part A Von Kossa Method for ALP Deposition and
Collagen Detection

• First select 2 of your 3 wells for staining and
  lysate
• - make sure these wells have plenty of cells
• - look for evidence of bone formation in 2 and 3
  week differentiated wells
• Follow protocol in lab manual
• - add solns in fume hood
• - do rinses at your bench
• Waste disposal
• - put liquid waste in marked containers at bench
• - put any pipet in contact with silver nitrate
  (steps 11-13) in marked container

• Dont forget to wear safety googles, gloves, and
  closed toed shoes
• Complete steps 1-4, then start part B
• - seal NFB fixed wells with parafilm
• - one partner can rinse off NFB while the other
  partner finishes part B
• Watch video during red violet staining (step 7)
• Step 16 air dry 5 min, then photograph cells
  and dispose of well plates

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Part B Making Osteoblast Lysates

• KEEP EVERYTHING COLD!!
• - put well plates on ice packs from freezer
• - put lysate tubes in chilling blocks from
  freezer
• Put correct lysate in designated colored tube
• Use cell culture PBS (it no longer needs to be
  sterile)
• - see Nell or Stephen if you need more PBS
• - label leftover PBS as non-sterile and
  replace in fridge
• Notes about scraping cells
• - start with undifferentiated well and work up
  to 3 wk differentiated well
• - you will have to work hard to scrap off
  mineralized bone

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Part C Observe Neuron Stamps

• Record observations in your lab notebook
• Take a picture of neurons adhered to coverslip
• Bleach plate, then use forceps to remove
  coverslips from well plate and toss in sharps bin
• Dispose of all neuron plates before leaving lab
  tonight (This is part of lab clean up!)

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Final Notes and Dates

• Lab Report 2 resubmissions due Thursday April
  24th at 1pm (graded reports returned at lab
  lecture this week)
• Final Exam Monday April 28th at 1pm
• - no labs that week
• - can have 1pg of notes
• Lab Report 3 due Monday May 5th at 5pm

• Before leaving lab tonight dump out ALL old cell
  media (this is part of lab clean-up)
• There are no pre-lab or post-lab exercises for
  next week, but dont forget to write protocol in
  your notebook
• - you do not need to include graphs
• - you should include charts and equations
• - leave space for data and calculations