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Jordan Garth Research Fall 2012

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Jordan Garth Research Fall 2012

  1. 1. U87MG-pe-GFP Cancer Cell Line in vitro Jordan Garth Undergraduate Research Fall 2012 Georgia Institute ofTechnology Bellamkonda Lab for Neuroengineering Mentor: Martha Betancur November 28, 2012
  2. 2. ResearchThis Semester  DARPA Project  Researched the failure mechanisms of neuronal electrodes  Sectioning  Staining  Imaging  Image Analysis  Eureka Project  ‘Exvading’ inoperable brain tumors in the cerebellum  Goal  to engineer a path of least resistance in using PCL nanofiber conduits for treatment
  3. 3. Using the U87MG-pe-GFP Cell Line  Why do we use U87MG-pe-GFP cancer cell line?  Able to inoculate in vivo and measure the tumor that develops  Common human glioblastoma cell line Tissue from an inoculated animal
  4. 4. Testing Ki67 and PCNA on U87 Cells  We cultured the U87 cells into the slides to test Ki-67 and PCNA  Ki-67 is a protein used to measure proliferation of the cells  Present in all cells life cycles except for G0  PCNA is a protein present during DNA synthesis  Goal  calculate proliferation rate in the cells
  5. 5. Cell Culture Overview  Incubate PS – 2 slides  Make complete media  Put frozen cells into the water broth  Cells adhere to the bottom  Washed with PBS, add trypsin to pick up the cells  Place in new media for an allotted time
  6. 6. Imaging Ki67 GFP DAPI Merged image of Ki67 10X (48 hr) Merged image of PCNA 10X (48 hr)
  7. 7. Imaging Continued Ki67 40X (48 hr) PCNA 40X (48 hr)
  8. 8. ImageAnalysis 0 10 20 30 40 50 60 70 Cell Count Dapi cell Count from Ki67 GFP cell Count from Ki67 Dapi cell Count from PCNA GFP cell Count from PCNA Average Cell Count 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Index (%cells not in G0) Ki67 index From Dapi Ki67 index from GFP PCNA index from Dapi PCNA index from GFP Percentage of Cells Proliferating Index = (Cells with proteins)/(Total cells detected with DAPI or GFP) N=9 images per staining
  9. 9. Future Studies  It is now clear to use Ki-67 to detect proliferation in U87MG- pe-GFP  Next step  use this stain in vivo sections  Get the proliferation rate using Ki-67 in U87MG-pe-GFP in the empty versus the aligned nanofilm conduits  I suspect that the proliferation rate of the cells is hypothesized to be less in vivo than in vitro  In vitro the cells have much more media to feed off of, whereas in vivo, the tumor cells are more starved
  10. 10. Questions?