WEEK ONE

- MONDAY 26/6/17 -

I was very nervous on the first day and wasn't sure what to expect.
Dr Tomlinson introduced me to the rest of the lab and gave me a quick lab induction, involving basic health & safety procedures.

The monoclonal antibodies (mAb) for Tspan14 need to be kept in the cold room.  I took them out briefly to pipette sodium azide into each of the samples.  This is important to prevent microbial contamination.

In the afternoon, I was allowed into the tissue culture room for the first time. I have had a few lectures, regarding cell culture techniques this year, so it was exciting to put this into practice for the first time. I watched as Ally (a PhD student) explained how to create an aseptic environment, how to prepare cell growth medium and the appropriate bins to dispose equipment afterwards.

We also counted the cells to determine whether there would be enough to do FACS using a haemocytomer.

- TUESDAY 27/6/17 - 

This morning I again watched Ally in the tissue culture room to learn about splitting and maintaining adherent cells.  We were working with the cell line HEK-293T, which doubles approximately every 24 hours.

When the cells are approximately 80% confluent (i.e. cover the entire plate with little space to grow), the cells will require splitting.  Splitting involves plating cells onto a new plate with fresh media. This ensures the cells grow healthily.  It is important to regularly check the status of your cells under a microscope to ensure cells aren't over-confluent.  Over-confluent cells may die or lose their phenotype.

In the afternoon, I watched Connie (a lab technician) as she developed her Western blot.  Western blotting is a routine technique for protein analysis, which uses antibodies to detect a specific antigen/protein.  The technique can generate quantitative and semi-quantitative data about the protein of interest.  To visualise the Western blot, traditionally the blot is developed on X-ray film in a dark room.  Connie used the Odyssey infrared imaging system instead of the traditional method because it is faster to use and creates a digital image that can be edited after as well.

 - WEDNESDAY 28/6/17 - 

Every Wednesday morning, the lab has a meeting.  It was interesting to see what everyone else had been working on and how the team shared ideas with each other.  Dr Tomlinson gave me 10 seconds to give a brief intro to what I would be doing as I don't have any data to present yet.

Today Ally showed me the technique for transfecting cells.  Before transfecting, I had to filter ~ 15ml of the Opti-MEM solution into a tube and placed it into a 37 degree water bath half an hour before transfection.

We had two plates of  HEK-293 cells.  One plate was transfected with an empty vector to be used as the control and the other with Tspan14 vector.  After, I had a go at splitting my own plate of HEK-293 cells into a 1:4 split, which was very exciting!

In the afternoon, I went into the tissue culture room with Dr Tomlinson to plate the hybridomas that had been sent with the Tspan14 monoclonal antibodies.  The hybridomas can either be frozen down for long-term storage or can be harvested for antibody production.  The hybridomas were plated into a small flask and grown in a "secret" media to maintain the cells.

- THURSDAY 29/6/17 - 

In the morning, I checked the hybridoma cells under the microscope with Dr Tomlinson to ensure the cells were growing nicely.  We also looked at the transfected and split cells from the previous day and decided that the transfected cells were ready for the experiment.

• Mix cells with mAbs for Tspan 14 - chose 8 different clones: 1A3, 1B6, 2H5, 3G3, 9D5, 9G1, 6E5, 10F3.

                                     o   +ve control = Tspan15 antibody: 5D4
                                     o   -ve  control = MOPC

• Secondary antibody = anti-mouse FITC (green fluor)

These samples were aliquoted into FACS tubes and had to be kept on ice

FACS
Dr Tomlinson introduced me to the FACS machine and went through how to set up the machine and connect it to the computer software - CellQuest Pro, which we were using to collect the data.

Summary of results 

• -ve control generated signal at approximately 10^1
• HEK293T cells with empty vector = low fluorescent signal because endogenous levels of Tspan14 are low and most of the Tspan14 is inside the cell.
• HEK293T cells transfected with Tspan14 = strong positive fluorescent signal.

Note: There was an issue with the FACS machine when collecting the data:
Very weak fluorescent signal when on low settings but dramatic increase in positive signal when turned up to high settings, which may have affected the data.  Differences in the positive peak in the FACS histograms may be due to differences in times when the high setting was switched on.

- FRIDAY 30/6/17 - 

In the morning, I had to analyse the FACS data collected on the previous day using the CellQuest Pro software.  The software allowed me to generate histograms of the data and overlay the graphs over the negative to show the positive shift with the Tspan 14 transfected cells.

Discussion of results

As expected the negative control generated a weak fluorescent signal at approximately 10^1.  By overlaying the FACS data for mock transfected cells with the negative control, they generated almost identical histograms.  This suggests that the antibody does not bind endogenous Tspan14, as it is expressed at low levels on the cell surface and most Tspan14 is found inside cells.  With the Tspan14 transfected cells, there surface proteins levels were overexpressed so the antibodies were able to bind generating a positive fluorescent peak shifted to the right of the negative control peak.  

There was also a shift in the fluorescence with Tspan15 antibody 5D4 in the transfected cells.  There may be a relationship between overexpression of Tspan14 and Tspan15 levels, that would be interesting to look into in the future (or the FACS machine might have just been playing up 😆).

In the afternoon, I attended a lab meeting for all members across the university within the "Molecules, Cells, Signalling and Health" research theme, where there were two talk given by a post-doc and PhD student.  It was very interesting to see what current research was being carried out in other labs.

After, I split my HEK-293 cells again to prepare them for transfection next week.  I did a 1:10 split onto two plates.