On Friday, Dr. Keese and I ran our first co-culture experiment with a 96 array! We used a 96w20idf, an array with interlocking pattern of electrodes. Because this is such a complicated experiment, Dr. Keese demonstrated the techniques needed for measuring out and pipetting our dilution series with the first 6 columns. He worked with the MDCK cells while I handled the BSC-I cells. It was helpful to get a refresher on how to passage cells, and how to handle 96 arrays. 

The first step was to get our cells to round up off the bottom of the flask using trypsin, and enzyme that temporarily damages cell walls (and also adding EDTA for the MDCK cells). Once the cells were  no longer a confluent layer, I added 20 mL of medium to the cells. I made sure the cells were evenly distributed throughout the solution by pipetting the mixture up and down, a technique known as titration. Then, I pipetted the cell-medium mixture into two tubes, and set those aside while I prepared the dilution series.

In this experiment, we were essentially running two separate dilution series: one with MDCK cells diluted with BSC-I cells, and the other with BSC-I diluted with MDCK. To recap the layout of this experiment, here are the tables I used in last week's post:

     

     Layout of cell ratios in the 96w20idf array: 

   
   Volumes of cells per column:

 

I labeled 5 tubes with each concentration ratio, and pipetted 4 mL of BSC-I cells into the first tube, 2mL into the second, then 1mL, .5mL, and finally, .25mL. Then, began to add in the MDCK cells that Dr. Keese had helped me passage. I did not put any into the first tube, as this will be pure BSC. The second tube I added 2ml, the third tube 3mL, then 3.5mL, and lastly 3.75mL. 

Now my dilution series is ready to be inserted into the wells of the array! After giving a quick flick to each tube to mix the cells around, I poured the first tube into a trough. Using the special, 8-tipped pipetter, I drew up enough liquid to fill each well (300 µL per well). I repeated this step 5 times, filling each column with a different BSC/MDCK cell mixture. Once Dr. Keese and I filled the entire array, it was off to the ECIS machine to plug in my array! Can't wait to see what this week's data looks like!

On Friday, I took a look at my data from my 8W1E co-culture experiment, dove deeper into the theory behind ECIS, and planned out my 96W20idf  experiment for next week.

ECIS Theory for Distribution:

Dr. Keese showed me a power point presentation that helped me understand the more technical side of ECIS. We spoke about the significance of different frequency levels and the physics behind the different types of 96-well arrays.

The first 96 array we spoke about is the 96W1E+, an array with two circular electrodes in each well. This array is used for measuring micromotion of cells, and for wound healing assays. Micromotion is the fluctuation of cell movement as measured by electrodes. Micromotion occurs even after a confluent layer of cells is formed, allowing us to take a close look at cell behavior throughout an entire experiment. 96W1E+ arrays are also used for wound healing assays. In a wound healing assay, electrodes send out electrical currents that will kill the cells in the vicinity of the electrode. This process will cause the cell membranes to become porous, and medium will leak into the cell, causing it to die. Wound healing assays allow us to examine the rate at which other cells will move in to heal the wound. 

96W1E+ with closeup on electrodes

The second array type we looked at was the 96W20idf array. This array has 96 wells, each with an interlocking pattern of electrodes which increases the total surface area significantly. More surface area allows us to take a look at more cells, therefore giving us data with greater statistical accuracy. However, for this accuracy we sacrifice the ability to examine micromotion and to perform wound-healing assays. Due to the large amount of electrode surface area, wounding would cause too many cells to die. I will be using this type of electrode in next week's experiment.

96W20idf with closeup on electrodes

Last week's data:

My data looked a little bit better than last week, yet I ended up with one outlier and my graphs seemed to suggest that my wells were a bit light on cells. Working with an 8 well electrode is much less statistically accurate because we are only looking at 2 wells of each cell mixture. If one (or both) of those wells differ from the true value, our data is more susceptible to error. Overall, I think my 96 experiments will be more  representative of what is really happening when MDCK and BSC-I cells are co-cultured. 

Next week's experiment:

I will be using a 96W20idf array to perform this co-culture experiment in a dilution series. It will be the most complicated experiment I've ever done, (and maybe even Applied Biophysics has ever done!) but here is the breakdown:

     Cells: BSC-I and MDCKII

     

     Layout: 

   
     Procedure: Using 10 tubes, we will dilute MDCK with BSC-I cells for the first five columns, and then dilute the BSC-I cells with MDCK in the last 5 columns. I will pipette 400 µl of cell mixture into each well. These will be the volumes of cells per column I will need for this experiment.