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Purdue Team Notebook

From May 6th - June 17th of 2009 The team researched possible topics. We knew we wanted to focus on cancer, but had to start from the beginning by researching as much as could before deciding on a final approach.

June 17th, 2009 Team has decided to focus on engineering microglia to recognize and label/destroy glioblastoma stem-like cancer cells.

June 18th - 23rd, 2009 The team has begun to search for reviews on microglia cells and to find potential markers for glioblastoma stem-like cancer cells.

June 24th, 2009 We have decided to use BV-2 microglial cell line. Have begun to search for labs in America that are using it to have a less expensive shipping cost. Also, created a gant chart. This will allow us to stay on top of our project, assess and meet our goals, and keep team members responsible for being apart of the project.

June 25th, 2009 Dr. Selkoe, from Harvard Medical School, has agreed to send us a sample of BV-2 cells. We are working to secure the correct media needed for both microglia and glioblastoma cells.

June 26th, 2009 The team has decided to meet on Fridays as well as Wednesdays to allow for more face-to-face communication.

June 29th, 2009 Today the team is having its first cell-culture meeting to prepare for working with mammalian cells. Today, we will send Dr. Selkoe our information to recieve the BV-2 line.

June 30th, 2009 The team will recieve a glioblastoma line from Dr. Yu who works with Cedars-Sinai Medical Center. We are meeting with a graduate student here at the university to discuss 3D culture.

July 1st - 20th, 2009 The team has been researching, buying media, finding protocols, and waiting for our cell lines to come in. Have begun inital designs of acutal constructs. Have chosen to go with two.

July 21st, 2009 The team recieved the BV-2 line from Harvard Medical School. Thanks again to Dr. Selkoe and Beth Ostaszewski for providing the line. We immediately plated the cells and will be checking on them in four hours. Our offical lab notebook has been started. We hope to get our glioblastoma line in this week or next.

July 22nd, 2009 Upon hearing the bad news that the glioblastoma line we were to recieve from Dr. Yu was completely gone, we researched papers looking for similar reagents used by other groups. Dr. Tofilon, from the Moffitt Cancer Center in Florida, has generously agreed to provide us with a vial of >90% CD133 cells. We are incredibly grateful to Dr. Tofilon and his students. We hope to have the rest of the media supply in by next week, and then recieve and begin working with the cells.

July 23rd, 2009 We have begun to grow up our BV-2s so that we can freeze them down. So far... so good. We hope to have our designed constructs done in two weeks and send them off to gene art. An expression vector has been picked out.

July 24th - August 28th, 2009 We've been culturing BV-2s and GAMB-1s...making sure we can keep them alive.

August 29th, 2009 Prepared 3D culture with BV-2 cells.

September 1st, 2009 Stained 3D cells to do a live/dead assay.

September 10th, 2009 Took fluorescent pictures of cells in 3D culture. They were mostly dead.  :(

September 22nd, 2009 Started growth rate experiment to determine doubling time for BV-2's in both BV-2 and GAMB1 media. Took preliminary photos (time = 0) at 2:25 pm.

September 23rd, 2009 Took 2nd data point for growth rate experiment. Took photos at 9:55 am (approx. 19 hours after plating).

September 30th, 2009 We presented our presentation to Indiana BioCrossRoads and a few memebers of biotech companies. We recieved great feedback and some very important questions to consider before we bring the project to MIT. We have begun to put in the final push before the October 21st deadline.

October 3rd, 2009 We began co-culturing the microglia and glioblastoma cells to see if there is any natural attractiveness. We were able to gain pictures at t3 due to having to wait 3 hours for the cells to settle. We did not want to create any reactions by jostling them. Migration was not able to be quantified, but we did learn that there is a natural attractiveness at t3. At t18, the attraction is slightly less. This may be due to a lack of chemotaxis coming from the stem-like cancer cells. Our designed construct was finally approved and sent to Genscript. Once we recieve it we hope to transfect and send it to MIT.

October 5th, 2009 We transformed the plasmid we recieved from Columbia to collect and purify the tat-p50-GFP fusion protein they provided us. We hope to use His-affinity columns to collect and then add it to the BV2 and GAMB1 to quantify uptake. This should contribute to our model and give us a better understanding of how the whole process will work. Currently waiting on the His column to come in. We have created a frozen stock.

October 8th, 2009 Worked on the model and maintained cell lines.

October 12th, 2009 Put the BV-2 and GAMB1 lines into a 3D pura matrix gel. This allowed us to see if the morphology of the GAMB1 would maintain a neuroshpere shape, and to redo the BV-2 experiment to make sure the first time was not a flook. We have t0 pictures as well. Due to scheduling conflicts we do not have fluorescent pictures, but we hope to have some for our power point presentation, as well, a co-culture in the 3D pura matirx.

October 17th, 2009 Brought up a new line of BV-2 from frozen stock to prepare them for transfection and maintained the GAMB1 line. No sign of the designed construct.

October 19th, 2009 No sign of construct or His-affinity columns. Have used this time to finish up the wiki and plan future experiments.

October 20th, 2009 Working on the wiki and have decided on our Halloween costume. Have been told that the construct will not be here until Friday :(

October 21st, 2009 Finishing up the wiki. Looking forward to seeing you all at the Jamboree!