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, 2014Objectives
Test ProceduresType I Collagen Test (Detection and Quantification)Collagen Staining Procedure & Microscope ImagingTrypsin Cell Detachment Procedure & Cell CountingTest ResultsCollagen Staining Procedure & Microscope Imaging Test
Trypsin Cell Detachment Procedure & Cell CountingPerformed on 3 scaffold layers.
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