User:Nancy T. Miles/Notebook/CHEM 572 Spr 2014/2014/04/22

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Objectives

  • Cell tests (only 2&3)

Test Procedures

Collagen Staining Procedure: Microscope Imaging and Collagen quantification

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

  • 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




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