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!