20.109(S08):Module 3
Module 3 Day 1: Start-up biomaterial engineering
Design/discussion day for students to choose parameters that will de-differentiate vs. promote phenotype of primary chondrocytes.
Module 3 Day 2: Initiate cell culture
Staff preps tissue digest day before; students prep 2D and 3D cultures (half-class at a time).
Other half of time can be diffusion modeling and NCBI bovine genome exploration exercises.
Note: 1 week between day 2 and day 3.
Module 3 Day 3: Testing cell viability
Take a portion of the cells, count and use Trypan exclusion, then prep for Live/Dead fluorescence assay and compare.
Module 3 Day 4: Preparing cells for analysis
Take portion of cells and isolate RNA, then run RT-PCR for collagen types I and II.
Rest of cells pellet with possible digestion step as well, depending on final protein assay chosen.
Meanwhile use ImageJ program to examine Live/Dead cells.
Module 3 Day 5: Transcript-level analysis
Run PCR fragments on gel and measure intensities using ImageJ.
Prepare day 1 of ?ELISA? (?Western?)
Module 3 Day 6: Protein-level analysis
Day 2 of collagen ELISA or Western.
If not possible, perhaps DMMB assay for proteoglycan instead.
