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

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==April 22, 2014==
==April 22, 2014==
===Objectives===
===Objectives===
*Cell tests
*Cell tests (only 2&3)


===Test Procedures===
===Test Procedures===
====Type I Collagen Test (Detection and Quantification)====
=====Type I Collagen Test (Detection and Quantification)=====
*[http://www.chondrex.com/products/mouse-type-i-collagen-detection-kit Company procedure ]
*[http://www.chondrex.com/products/mouse-type-i-collagen-detection-kit Company procedure ]


====Collagen Staining Procedure & Microscope Imaging====
=====Collagen Staining Procedure & Microscope Imaging=====
*[http://www.chondrex.com/products/sirius-red-fast-green-collagen-staining-kit Company procedure]
*[http://www.chondrex.com/products/sirius-red-fast-green-collagen-staining-kit Company procedure]


====Trypsin Cell Detachment Procedure & Cell Counting====
=====Trypsin Cell Detachment Procedure & Cell Counting=====
*[http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/1/celldetachmentprotocol.pdf Company procedure]
*[http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/1/celldetachmentprotocol.pdf Company procedure]


===Test Results===
=====Collagen Staining Procedure & Microscope Imaging Test=====
*Absorbance data:
**Original
**pD only
**pD & original
**Brute force
**Plain


*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
***Original
***pD only
***pD & original
***Brute force
***Plain
*Microscope images?
=====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


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Revision as of 12:21, 22 April 2014

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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:
    • Original
    • pD only
    • pD & original
    • Brute force
    • Plain
  • 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
      • Original
      • pD only
      • pD & original
      • Brute force
      • Plain
  • Microscope images?
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


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