WEEK EIGHT ~ my last week! :(

- MONDAY 14/8/17 -

This week I will redo the FACS experiments, (1) with the Tspan14 cholesterol mutants and (2) with the ADAM10 KO cells.

Today, I split the HEK WT and HEK T14 + C3 cells.  I also seeded 4 plates with1 x 10^6 cells in 3ml media in each for transfection tomorrow. 

                                        - TUESDAY 15/8/17 -
Today I transfected the cells with the appropriate plasmid DNA as follows:
                                         (1) pEF6.C - His

                          (2) pEF6.C FLAG human Tspan14

                                (3) Tspan14 Q to A mutant

                                 (4) Tspan14 Q247E mutant 

- WEDNESDAY 16/8/17 -

The day started with my last lab meeting and I presented the data from the Tspan14 cholesterol mutant FACS repeat and the ADAM10 KO FACS data. Whilst it looks like 6E5 and 10F3 could have been recognising Tspan14 in the ADAM10 KO cells - when Ally did the experiments the shifts were very small.  I will have to redo the experiment to see if the data agrees.

For the ADAM10 KO experiment, I used HEK cells stably transfected with Tspan14 as a positive control. However the shifts were not as significant as seen in previous experiments. Connie suggested that  because I've previously tested the antibodies with HEK cells transiently transfected with hTspan14, the data would be different with stably transfected cells. In the transiently transfected cells there are varying levels of Tspan14.  In the stably transfected cells, the C3 clone has one of the highest expression levels of Tspan14, so it is strange that the staining is a lot weaker than in the transiently transfected cells. 

In the afternoon, I repeated the FACS experiment with the cholesterol mutants. The antibodies recognised the Q to A mutants, which agrees with the data from last week. However, they also recognise the less harsh mutation Q247E, which suggests that the previous transfection didn't work very well. 

After running the FACS, I did a 1:5 split of my cells.  I also did a 1:2 split of the Tspan14 stably transfected cells for the FACS experiment tomorrow. 

- THURSDAY 17/8/17 -

Today, I'm repeating the FACS experiment with the ADAM10 KO cells. 

The FACS data (below) agrees with the previous experiment (Week 7 - Friday) as the only two antibodies that recognised Tspan14 on ADAM10 KO cells were 6E5 and 10F3.  This may be suggesting that 6E5 and 10F3 are recognising an epitope that is masked by ADAM10.