96-well seeding

Hello all you wonderful people! I hope you had a great week – I certainly did! This week is more of a continuation of the previous week, so I’m hoping you will be just as intrigued as last week!

Before we go into that, I just wanted to show you guys some pictures of my cells!

So this is a picture of the cells at about 70% confluency – they are healthy and they have space for a day or two before they must be passaged.

Now, onto the good stuff! As promised, in this post, I will be going into detail about seeding the 96 well. Taking a step back to the previous blog (before we made the new flask), I mentioned something about adding 4.5 mL of medium to cells after adding trypsin to them (to dissociate them). After this, I resuspended the cells by repeatedly sucking them in and out. Now, here comes the new stuff. After resuspension, I take about 200 microliters of the solution and put it into a small tube.

Next, I have to count the cells – but if I need to count all of them by hand or by eye (which would mean that I have superpower vision that every optometrist dreams of), I would have grey hair by the end of it. Thus, I will be using a machine that simplifies the whole process. So first, I took a strip of parafilm, and then I pipetted 10 microliters of trypan blue (the dye the machine uses) as a drop onto the parafilm. Next, I resuspended the cells again (from the small tube from the previous step) – because cells have a tendency to drop to the bottom, so resuspending will help them be well-dispersed throughout. Next, I took 10 microliters of the cell solution and put it into the drop of dye, and then resuspended it a couple of times. Then, I took 10 microliters of this new mixed drop, and then put it into the machine. Then, all I had to do was press the button that said count – and I was good to go!

The machine gives a whole bunch of numbers – but the ones I really care about are the viability (percentage of healthy cells) and the live cell count. For this particular cell line, A549, anything above a 95% viability is pretty good – and luckily, all of mine have been above 96 when I counted them (which is definitely a good thing 🙂)

Now, onto 96-well seeding! So we use the live cell count number to do some calculations. Essentially, after discussing this with my mentor, we decided that 3000 cells per well (which is 100 microliters per well) is a good number to have – however, the number of cells from the cell count number is way too high. Thus, we need to dilute it, in a sense.

So this is the excel sheet (thank heavens for excel sheets) I made that details all the calculations. Essentially, the dilution factor is obtained by dividing the total live cells (calculated by the machine) by the cells/ml number (the cell concentration the counted cell concentration needs to be diluted to). For total volume, we need 96 wells x 100 microliter/well = 9600 microliters = 9.6 mL, which rounds up to about 10 mL. To find the amount of cell solution we need, divide total volume needed by the dilution factor, and then to find the amount of medium needed, subtract total volume from the cell volume – and BINGO, you have everything you need to create a seeding solution.

After pipetting the right amounts of medium and cell solutions into a tube, I then resuspended the solutions just to mix it up well to ensure the cells are evenly dispersed. Next, I used a micropipette to pipette 100 microliters of this seeding solution into every single well. It actually takes a lot of concentration and good pipetting technique (which sadly, took a long time for me to even start developing). Then, I incubated the 96 well.

This 96-well is what I will be putting into the proliferation assay to conduct my experiment. But, more on that later! In the next post, I hope to talk more about the treatment and the proliferation as well. I hope you have a great rest of the week!