U87MG-pe-GFP Cancer Cell Line in vitro

Jordan GarthUndergraduate Research Fall 2012

Georgia Institute of TechnologyBellamkonda Lab for Neuroengineering

Mentor: Martha BetancurNovember 28, 2012

Research This 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

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

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

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

Imaging

Ki67

GFP

DAPI

Merged image of Ki67 10X (48 hr)

Merged image of PCNA 10X (48 hr)

Imaging Continued

Ki67 40X (48 hr) PCNA 40X (48 hr)

Image Analysis

Cell Count0

10

20

30

40

50

60

70

Dapi cell Count from Ki67 GFP cell Count from Ki67Dapi cell Count from PCNAGFP cell Count from PCNA

Average Cell Count

Index (%cells not in G0) 0%

10%

20%

30%

40%

50%

60%

70%

Ki67 index From DapiKi67 index from GFPPCNA index from DapiPCNA index from GFP

Percentage of Cells Proliferating

Index = (Cells with proteins)/(Total cells detected with DAPI or GFP)

N=9 images per staining

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

Questions?