Negative isolation of CD4+ T cells

Recently I've begun using a flow cytometry sorter to isolate the Treg populations I'm most interested in looking at. However I've been having a tough time getting the relatively high number of cells that I need considering that Tregs make up such as small percentage of CD4+ T cells (roughly 10%). It's not feasible (as I've found out) to spend 6 hours collecting cells for one group and then have another 6 hour run immediately after for the control group. I've made attempts at enriching the Treg population before sorting with little success. Luckily for me, I've just come across a new product that I am hopeful will be more helpful! In order to determine if this enrichment is worth doing I directly compared it to our current product usage and protocol.

 The above video provides a nice cartoon demonstration of how their magnetic separation works along with a brief demonstration of the protocol. 

This video is just funny so I decided to include it, even though it doesn't provide any extra information. Talk about knowing your audience! 

We were given a free sample of the EasySep™ Mouse CD4+ T Cell Isolation Kit  from Stemcell as well as one of their magnets to try it out. 

The Experiment: 

2 C57Bl/6 spleens ( mice were 12 weeks old)
competitor's column based kit

EasySep™ Mouse CD4+ T Cell Isolation Kit (sample kit our lab got from the company)

1. Disrupt the spleens mechanically to create a uniform cell suspension.
2. Resuspend cells in the cell media recommended for each kit.
3. Perform the isolation. Each kit used its own magnetic set up.

I used a sample for the "before" plot after I'd added the magnetic beads to each. That is why the cell numbers are different for each of the first plots. The second and third plots in each row are replicates.

Top row: Competitor's kit. The first plot is the "before" isolation and the next two are replicates of the "after" samples. Bottom row: EasySep™ Mouse CD4+ T Cell Isolation Kit. The first plot is the "before" and the next two are replicates of the "after samples.

As you can see from the data, the EasySep kit worked the best. I believe we might have received a "bad" batch of beads for the competitor's kit. As I'd already used this kit twice before and gotten similar results, I'm confident that this result isn't a once off for the beads we received. Results like these are the reason I started looking for a new kit in the first place. In addition it takes me about 1.5 hours to prepare 5 spleens worth of cells for the competitor's kit. The EasySep kit literally took 20 mins tops. I'm sold on the new stuff!

...and we're back!

The first entry I wrote for this blog was nearly 5 years ago and there it sat completely forgotten as life moved on. Since then I've worked in more labs, traveled to more states and started my graduate studies in pursuit of a Ph.D. at the University of Washington. I hope to use this space not only to keep myself motivated but to stoke the fires of my own curiosity which feel as if they are being slowly snuffed out by the myriad scientific failures that have dogged me these last nearly 12 months.

Primarily I hope to use this as a repository for (hopefully) progressively better summaries and thoughts on papers that I read. I hope to have 2 or 3 done a week and if I succeed to increase the frequency to one a day. However, baby steps are a must. Onward and upward!!