1/3/17: A Brief Summery of My Day

I went to my internship over winter break and ran the cell swelling assay (check out my previous post) and I will continue to go to my internship over my school's Intersession. This was my first day back to lab after my mini vacation and there was a lot to do. I first started my day off thawing cells. Since cells need constant care, the cells were frozen before Christmas break. After we got back, they were thawed and hopefully will be confluent in about a week. The process for cell thawing is as follows:

1. Take the cells out of the freezer and place in water bath briefly

2. Remove the tube or container from the water bath, its okay if it still has some ice in it.

3. Add the cells to the flasks prefilled with media.

These steps should be done quickly because it is important to remove the cells from the concentrated Dimethyl sulfoxide (DMSO). DMSO is used to prevent ice crystal forming inside cell. At high concentrations and in room temperature, DMSO is toxic.

After thawing cells, I helped image cross section of mouse hearts. The mice were injected with isoproterenol, a drug that stimulates the same receptors as adrenaline. In previous studies, it was found that this drug increased heart size and induced fibrosis or the build up of collagen. In this experiment, Jon was looking at the cardiac hypertrophy and fibrosis between wild type mice and mice without the CMH gene.

Following the picture taking, Jon and I worked on a protocol that stained the nuclei of cells using a light microscope. Jon believes that the progeria patient with the caveolin mutation has a deformed nucleus. The following goes over how cells are stained.

1. Cells are fixated, so all the proteins become linked so that nothing moves.

2. Cell are treated with 0.5% triton for ten to fifteen minutes

3. Wash or rinse cells with PBS three times and add 3% BSA stock (blocker)

4. Place the cells on a rocking plate and wait for about an hour

5. Rinse and add primary antibody, leave over night.

6. Rinse and then add secondary antibody

7. Image!

So first the cells are fixated so then none of the proteins can move around, they are all covalently bonded to each other in a complex pattern. Then, the triton permeabilizes the cell, which makes it more likely to let statins and dyes go into it. Jon was planning on staining caveolin by using antibodies that specifically bind to it. But first, the cell must be blocked with BSA solution. BSA weakly binds to all the proteins inside the cell. This way when the primary antibody is added, it doesn't weakly bind to all proteins, instead, it strongly binds to one protein (caveolin). After the primary is added, then a more general secondary antibody is added to the plate. The secondary antibody is more general in that it binds to a specific animal’s antibody. The secondary antibody is fluorescent. The amount of light emitted from the sample is supposed to represent the amount of caveolin.