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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Cell Tests</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Cell Tests</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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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===
===Test Results===
====Type I Collagen Test====
=====Collagen Staining Procedure & Microscope Imaging Test=====
*Absorbance data:
[[Image:2014_0422_abs_collagen_quantification_test.PNG]]


====Trypsin Cell Detachment Procedure & Cell Counting====
*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
[[Image:2014_0422_collagen_quantification_calcs.png]]
 
=====Trypsin Cell Detachment Procedure & Cell Counting=====
Performed on 3 scaffold layers.
*“Original” - 125 cells on scaffold
*“Original” - 125 cells on scaffold
*Polydopmaine & “original” - 450 cells on scaffold
*Polydopmaine & “original” - 450 cells on scaffold
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===Next time===  
===Next time===  
*Perform all tests in triplicate
*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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Latest revision as of 23:54, 26 September 2017

Cell Tests Main project page
Previous entry      Next entry

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:

  • 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


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