Time Lapse Attempt #1

Today, Dr. Keese, Dr. Renken and I set up the equipment required for our time lapse imaging of experiment. In this experiment we will be examining how BSC-1 and MDCK cells interact with one another when co-cultred. As we have seen in the past, MDCK cells have the tendency to form like-cell islands amongst BSC-1 cells. We would like to see how these islands are formed over time. The basic mechanisms required for our time-lapse experiment are:

• Phase contrast microscope
• special ECIS loading dock with windows, water channel system, and gas inlet
• BSC-1 and MDCK cells
• CO2 pump and bubbler
• Nixon camera with microscope lens attachment
• Water pump 

Here is an image of the time lapse setup:

Most of the equipment used in this setup is to keep the cells healthy & happy. Cells like to stay in a warm, moist, environment- hence the water pump that sends 37 degree Celsius water through the channels of the dock they are encased in. The CO2 pump keeps the cells at ~ 5% CO2. The rest of the setup is pretty self explanatory: the camera is set up to take 1,000 frames over a 72 hour period, and the microscope is for magnifying the cells using phase contrast. Phase contrast is essentially a system of lenses that are arranged to allow a specific amount of light in, thus highlighting cells which are otherwise transparent bodies. Here is a picture that demonstrates how phase contrast highlights a cell:

Cells magnified with traditional bright field microscope (left) and phase contrast (right).

In Friday's experiment, we are just looking a BSC-1 cells using time lapse. This test run will help us work out any kinks such as adjusting the magnification, CO2 levels, etc. in order to be prepared for this Friday when we place a co-cultured sample of BSC-1 cells and MDCK cells into this contraption! I'm not sure if we are going to add ECIS measurements to this Friday's run, although that is possible for us to do. I'm hoping our experiment went well and I'll be able to post a time-lapse movie on my next blog post!

Hu Tu-80 Dilution Series and L-cell Islands

Today at Applied Biophysics I ran an experiment using a new type of cell known as Hu Tu-80. Despite its catchy sounding name, Hu Tu-80 cells are actually a bit sinister- they are human intestinal cancer cells. So far I have worked only with animal cells- from monkeys, dogs, and mice. This new human cell line looked similar to the animal cells I have worked with. Hu Tu-80 cells share some of the same properties as MDCK cells such as their cobblestone-shaped growth, but had more defined cell boundaries similar to that of BSC-1 cells.

This experiment is designed to examine the effects of BSA and Gelatin on varying concentrations of Hu Tu-80 cells. In this experiment, I used a 96W1E+ (which is an electrode array with 96 wells, and one electrode in each well). In the odd columns, I coated the wells with BSA (Bovine Serum Albumin), and the even columns were coated with Gelatin. BSA is used as a protective agent for cells from oxidative damage, and also serves to stabilize components of media such as fatty acids and pyridoxal. Gelatin is made from the collagen of animal parts and is primarily used to improve cell attachment. I let 200 micro liters of these two solutions sit in the wells for about 5 minutes, suctioned out the wells, and then added varying concentrations of Hu Tu-80 cell mixture to each of the wells. The Hu Tu-80 cell solution was diluted with a corresponding amount of regular medium. Here is the layout of my 96 array (the last two columns are empty):

Green columns are BSA coated, yellow columns are Gelatin coated.

L-cell and BSC-1 dilution series results:

Two weeks ago, I performed a dilution series experiment with L-cells and BSC-1 cells. I was testing these two cells types to see if they formed islands like the BSC-1 and MDCK cells did. The result: YES! As the concentration of L-cells increased, L-cell islands became more prevalent. Dr. Keese stained a row of my array to show how the number of islands increased. In the following pictures, you can see these stained wells:    ( *L-cells are the dark, circular cells* )

Pure BSC-1

Light inoculation - .0625 concentration of L cells

L-cells islands become more prevalent at .25 concentration

L-cell islands become as populous as BSC-1 cells

Pure L-cells

Next week, I will be working with Dr. Keese to begin setting up the time lapse equipment for our next experiment! We will be using the time lapse video to watch the formation of these islands in real time. Can't wait!!

L-Cell dilution series and hemocytometer usage

After a two-week vacation, it was refreshing to get back to my internship at Applied Biophysics. It was great to get back to the lab, creating a new co-culturing experiment and learning learning how to count cells using a Hemocytometer.

Before I delve into the details of my internship on Friday, I'd like to write a quick happy birthday to the amazing Dr. Ivar Giaever who celebrated his 85th birthday on Saturday! An esteemed physicist, Dr. Ivar Giaever is a professor emeritus at RPI, a professor-at-large at the University of Oslo, and president of Applied Biophysics. And did I mention he won the 1973 Nobel Prize in Physics for his studies in tunneling phenomena in superconductors? I love hearing stories about Dr. Giaever, from his stories of his travels to across the world, to his take on global climate change. Happy birthday!

Back to the science! I am now working on another dilution series that has the same format as my past 96w20idf experiments with MDCK and BSC-1 cells, but instead of MDCK, I am using L-cells (mouse endothelial cells). The object of this experiment is to compare the behaviors of our previous cell combinations of MDCK and BSC-1 (epithelial/epithelial) to a mixture of BSC-1 and L-Cells (epithelial/fibroblast). Here is the layout of this 96w20idf dilution series:

Cell Counting using Hemocytometer:

Once I finished setting up my dilution series experiment, I learned how to use a hemocytometer to manually count cells. The idea behind this device is to take a small, manageable amount sample of cell mixture, and use it to make an estimate of how many cells are present in a larger volume of that same mixture. Hemocytometers are commonly used to count blood cells (hence the name hemo (meaning blood) cyto (meaning cell) and meter (meaning count)). Here is what a hemocytometer looks like:

To use the hemocytometer, one pipettes a small drop of cells suspended in medium into the tiny v-shaped notch as seen in the image above. There is a cover slide that sits on top of the raised part of the hemocytometer, which the cell solution slips under and fills. Capillary action draws the liquid into the space between the cover slip and the glass surface, creating a uniform layer of liquid .1mm in depth. There is a microscopic, laser-etched grid on both sides of the hemocytometer, which looks like this:

Using the applicable objective, I magnified one of the 4x4 squares, and counted the number of cells within this square. With cells that are on the edge of the square, only count those that touch the top and left sides of the square (it's just common practice). To calculate the cell concentration per ml, use this simple formula:

I counted up 5 of the 4x4 squares for both a sample of L-cells and a sample of BSC-1 cells which were left over from my dilution series experiment. I counted up 32 L cells and 34 BSC-1 cells, two values that are relatively similar. This similarity is good because that means the cell solutions used in my dilution series will be relatively reliable. 

We did not perform this in my experiment, but one can use a hemocytometer to calculate the cell viability count by staining cells with Trypan Blue. Trypan Blue is a "vital stain" that colors dead cells  blue due to the incorporation of color into the proteins of dead cells, and leaves live cells colorless.