User:Madeleine Y. Bee/Notebook/CHEM-572 2014S/2014/04/22

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April 22, 2014

Objectives

  • Cell tests (only 2&3)

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
  • Absorbance data:

2014 0422 abs collagen quantification test.PNG

  • 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

2014 0422 collagen quantification calcs.png

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