WEEK SIX

- MONDAY 31/7/17 -

Today, I did a 1:5 split of my cells.  I also seeded HEK T14 cells onto two coverslips so that I could do a repeat with 9D5 (since the last coverslip snapped in half and had no cells on!) 

In the afternoon, I went to a meeting with Connie and her two sueprvisors: Dr Tomlinson and Dr Natalie Poulter.  It was very interesting to hear about Dr Tomlinson's ideas for future projects including how ADAM10 could be used as a drug target to treat cancers. 

Discussion of my project so far: 

2H5 is "dead"

Dr Tomlinson explained that it is normal for monoclonal antibodies to become unstable and degrade. However, we still have frozen vials of the hybridoma cells that can be used to rescue the antibody. For now, I will no longer be using 2H5 in my experiments (less tubes for FACS!!! 😀🎉)

IS TSPAN14 MOSTLY INTRACELLULAR?
We also talked about permeabilizing the cell membrane and doing intracellular staining for analysis by FACS and microscopy.  This will then help determine whether most of the proteins are intracellular and would explain why the antibodies haven't been recognising endogenous Tspan14 on the cell surface. 

ARE THE EPITOPES BEING BLOCKED BY ADAM10?
Most of the Tspan14 on the cell could be interacting with its partner protein ADAM10. This interaction may be masking the key epitopes for the antibody to bind. Therefore, it would be interesting to do a FACS experiment with ADAM10 knockout cells. 

COULD 9G1 BE DOING SOMETHING INTERESTING HERE? 

All the antibodies recognised the main extracellular region (EC2) apart from 9G1 and 2H5. 

We already know that 2H5 is unstable and 9G1 was still recognising overexpressed Tspan14. Dr Tomlinson suggested that perhaps 9G1 specifically recognises the small extracellular region, or specific open/closed conformation of Tspan14.  

The meeting was very useful and helped me relate my data and what I've been doing in the lab back to the theory.  The next steps for my project are: 

(1) Intracellular FACS
(2) FACS with ADAM10 KO cells
(3) FACS with cholesterol mutants
(4) Redo FACS with CD9/Tspan14 EC2 mutant
(5) On Wednesday, we'll go back to the microscope to image the WT cells again at a higher intensity and compare the signal to that of the MOPC control to see if the antibodies can recognise endogenous Tspan14 by confocal microscopy. 

- TUESDAY 1/8/17 - (August is here already!)

Today, I checked the two coverslips and there seemed to be more cells on them compared to last week, which was a good start! I then fixed and stained the two coverslips with: 

(1) Primary antibody = 9D5
(2) Secondary antibody = anti-mouse antibody raised in goat and conjugated with Alexa488

Alexa fluor 488 is a green fluorescent dye. it fluoresces specifically in the laser line with wavelength 488nm

In the afternoon, I downloaded  the Fiji software (which stands for "Fiji is just ImageJ") to add scale bars to the images and change them to grayscale. 

MOPC with increasing gradient of intensity

There wasn't any signal with the WT cells at 6%. There was some signal at higher intensities, but this could have mostly been background noise rather than a true signal. 

So on Wednesday, Connie has booked another imaging session to see if there is any signal at higher intensities. 

2H5 didn't produce any signal, asexpected.

1A3 might not recognise Tspan14 on fixed cells therefore didn't produce any signal.  

All the other antibodies produced a signal. However, there was variability in the strength of the signals.  This may be because there were different numbers of cells on each coverslip? 

- WEDNESDAY 2/8/17 - 

This morning, we had a lab meeting to catch up with Dr Tomlinson since he has been away for 3 weeks. He gave us a brief overview of some of the talks from the conference he attended.  It was nice to also hear what everyone else had been up to in the lab. 

I seeded 4 plates of HEK WT cells for transfection tomorrow and did a 1:5 split of the WT and HEK T14 cells as well. 

In the afternoon, I went to the med school to image the WT cells again at a higher intensity and also to see if 9D5 gave any signal with the overexpressing cells.  However, the microscope decided to die and neither Connie or Natalie could fix it so we decided to leave the imaging for now (so the microscope doesn't like me either). 

- THURSDAY 3/8/17 -

waiting for DNA/PEI complexes to form

Transfected the cells 

Today I transfected the HEK cells with the following constructs

(1) pEF6.C His

 (2) pEF6 FLAG human T14    (3) pEF6.C Tspan14 A mut

      (4) pEF6.C T14 Q247E

- FRIDAY 4/8/17 -

Today I did the FACS experiment to see if the antibodies can recognise the open/closed conformation of Tspan14 using two Tspan14 cholesterol mutants: (1) pEF6.C Tspan14 A mut and (2) pEF6.C Tspan14 Q247E.

The first is the true mutant.  This mutation causes the tetraspanin to remain in its open conformation so no cholesterol can bind.  It is thought that this allows better binding and trafficking of ADAM10.  The second mutant is a glutamine to glutamate mutation at position 247.  This mutation is less harsh and so cholesterol is still able to bind. 

The FACS data generated shows that all the antibodies recognised both mutants.  There was stronger recognition with the true mutant than with the Q247E mutant.