April 22, 2014
Objectives
Test Procedures
Type I Collagen Test (Detection and Quantification)
Collagen Staining Procedure & Microscope Imaging
Trypsin Cell Detachment Procedure & Cell Counting
Test Results
Collagen Staining Procedure & Microscope Imaging Test
- Calculations (from company procedure)
- "To calculate the amount of collagen, correct the OD 540 value by subtracting the contribution of Fast Freen at 540nm, which is 29.1% of the OD 605 value. The color equivalence (OD values/ug protein) is 0.0378 for collagen and 0.00204 for non-collagenous protein at OD 540 and 605, respectively."
- Collagen (ug/3 scaffold layers)= [OD 540 value-(OD 605 value x 0.291)]/0.0378
- Non-collagenous proteins (ug/3 scaffold layers)= OD 605 value/0.00204
Trypsin Cell Detachment Procedure & Cell Counting
Performed on 3 scaffold layers.
- “Original” - 125 cells on scaffold
- Polydopmaine & “original” - 450 cells on scaffold
- Polydopmaine only- 150 cells on scaffold
- Brute force - 300 cells on scaffold
- Plain - 100 cells on scaffold
Next time
- Perform all three tests in triplicate
- Have one scaffold for assays that has no cells one it (for blank & with "leftover" media [under microscope?])
- Keep solutions post-assay to look at under microscope to see if cells are still there
|