User:Nancy T. Miles/Notebook/CHEM 572 Spr 2014/2014/04/22: Difference between revisions
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|style="background-color: #EEE"|[[Image: | |style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> Biomaterials Design Lab</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> </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"|<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> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
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== | |||
* | ==Objectives== | ||
*Cell tests (only 2&3) | |||
==Test Procedures== | |||
====Collagen Staining Procedure: Microscope Imaging and Collagen quantification==== | |||
*[http://www.chondrex.com/products/sirius-red-fast-green-collagen-staining-kit Company procedure] | |||
=====Collagen staining===== | |||
*The cells were cultured in a 3-inch petri dish, each dish initially containing approximately 3x104 cells | |||
*Each dish included three 3-layered scaffolds that underwent different treatments | |||
*When immersed in Kahle fixative, the protocol called for 0.5 mL of solution, but for the purpose of this experiment, more Kahle fixative solution, about 4 mL, was used to immerse the scaffold | |||
*The fixative was removed after 10 minutes of incubation at room temperature and washed with PBS, and enough Dye Solution, about 1 mL, was added to each dish and scaffold | |||
*The cells were incubated for 30 minutes, as directed. The dishes were rinsed with distilled water 4 times before visualizing the scaffolds in culture under the microscope | |||
*One scaffold from each plate was removed and placed into a new dish to examine the scaffolds alone | |||
=====Quantification===== | |||
*To extract the dye from cell culture for quantification, 2 mL of Dye extraction buffer was added to each petri dish containing just the scaffolds, no cells, and pipetted up and down to extract the dye from scaffolds | |||
*The dye solution was then collected and placed in the spectrophotometer for quantification of collagen and non-collagenous proteins | |||
**The Sirius red stained collagen had an absorption at 540 nm while non-collagenous materials, stained Fast Green, had an absorption at 605 nm. | |||
====Trypsin Cell Detachment Procedure & Cell Counting==== | |||
*[http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/1/celldetachmentprotocol.pdf Company procedure] | |||
*The cells were cultured in 3-inch petri dishes overnight, each well initially containing approximately 3x104 cells with three 3-layered scaffolds that underwent different treatments | |||
*The cell culture media was removed, and the scaffolds were placed in new petri dishes | |||
*They were trypsinized with 1 mL of trypsin for 5 minutes. Excess trypsin was removed from the plate very carefully, so as not to disrupt the cells on the scaffolds, and the scaffolds were incubated for another 5 minutes | |||
*3 mL of growth medium was added to each plate and pipetted to suspend cells in media | |||
*The cells in media were counted using a cell counter. The quantities of cells were compared among different scaffold/nanofiber treatments. | |||
==Test Results== | |||
====Collagen Staining Procedure & Microscope Imaging Test==== | |||
*Absorbance data: | |||
[[Image: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 | |||
[[Image: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 | |||
Revision as of 09:07, 7 May 2014
Biomaterials Design Lab | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Objectives
Test ProceduresCollagen Staining Procedure: Microscope Imaging and Collagen quantificationCollagen staining
Quantification
Trypsin Cell Detachment Procedure & Cell Counting
Test ResultsCollagen Staining Procedure & Microscope Imaging Test
Trypsin Cell Detachment Procedure & Cell CountingPerformed on 3 scaffold layers.
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