A List of Solutions For The Cell Swelling Assay

After my run last Friday (12/21/17), I decided to learn more about the solutions I will be working with.

H2O2:

In a previous experiment, it was found that when mouse myoblasts were treated with H202 prior to undergoing the hypo-osmotic treatment, there was greater cell death. They think this has to do with how caveolin-1 is broken down in the presence of H2O2. Caveolin-1 is also necessary for the formation of caveolae, which help regulate and maintain cell shape in hypo-osmotic environments. They showed that H2O2 had similar effects in decreasing caveolae effectiveness as methyl-beta-cyclodextrin and siRNA that specifically blocked caveolae function.

Trypan Blue:

Trypan blue is a negatively charged dye that cannot permeabilize an alive cell. This is because the inside of the cell is slightly negative and the plasma membrane does not contain a specific protein that can serve as a pathway that would allow Trypan blue to go inside. When a cell dies, the bilipid membrane is compromised and the dye can enter the cell.

Hypo-osmotic media:

30 osm media is used for the experiment. Hypo-osmotic media is an effective way to measure caveolae, little invaginations of the plasma membrane, function because they are the main regulators of cell shape in hypo-osmotic environments (c2c12 people cite).

MEM media:

MEM media is used to grow human dermal fibroblasts. MEM media contains amino acids, salts (calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, and monosodium phosphate), glucose, and vitamins (folic acid, nicotinamide, riboflavin, B12). It also contains sodium bicarbonate to maintain the ideal pH for cell growth. The problem with MEM media and the bicarbonate buffer system is that bicarbonate pKa value is around 6.1. In the presence of CO2, the bicarbonate buffer is able to maintain a pH between 7.2 - 7.6, the optimal range for cell growth. For this system to work, there needs to be higher concentration of CO2 than what the cells produce as a by product. Bicarbonate buffers do not work in normal atmospheric conditions.

It was found that the pH of the buffer could change rapidly in the time span and cause cell death. To prevent this, in the future, HEPES and HBSS will be used.

HEPES:

Is a salt buffer that can maintain pH between 7.2 - 7.6 in at room conditions. One downside of this buffer is the production of H2O2 and other reactive oxygen. This can be prevented if the samples are stored in a dark room or in aluminum foil or if 2 mM of sodium pyruvate is added (check fact).

HBSS:

Hank’s buffered salt saline is a buffer that maintains the correct pH and osmolarity for cells. It is usually used like PBS. It is used to clean any containers that will come in contact with cells. HBSS is also used to rinse cells.