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. 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. 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. 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. 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
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. 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
U87 cells are cancer cells that are modified by Dr. Pai attached plasmid (codes for GFP) that the cells took it in and produced GFP. We are able to identify them. I helped with the in vitro cell line. Moving on this is what I will be working on
They were stained for a specific proteins that denote antibodies/proteins that indicate inflammation. Imaged the results of the staining. Image analysis to measure the inflammatory factors.
Able to enoculate an animal and measure the tumor that develops
Pretty simple since they are cancer cells. 2 slides to stain and image. We want to measure all the proteins in the
Index = Cells with proteins/Total cells detected (with dapi or gfp)
Now use the Ki67 in the in vivo tissue and get the proliferation in the Ki67
