The road ahead...

I successfully navigated my first committee meeting a couple of months ago and now it's time to lay out a concrete plan for the next 5 months. I need to ensure that I will be the most prepared I can be for the general exam. So...the hunt is on for some tools to help me accomplish that goal! 

Namely I'm looking for a couple of different classes of tools: a planning program, a project scheduler and some kind of resource manager to help me keep on top of the reading I need to do. I would prefer to use free programs and tools as I'm on a pretty tight budget however I recognize that programmers and developers have to eat too. 

I'll keep this spot open for updates on the three categories. Stay tuned! 

Rock out with your pipet out!

The last few weeks have been a bit of a struggle as I and my fellow lab mates try to push back the frontiers of science. A noble undertaking indeed! However every science warrior eventually starts to wear down and even the most chipper amongst us starts to sag a little bit at the corners of the mouth after the 8th hour of experiment preparation and mindless pipetting.

What's an enterprising and determined lab mouse to do you ask?

Let me introduce to you, Music(TM) the fix-it for all repetitive lab tasks! Enough with the tomfoolery, I've had my fun. In all seriousness, we've just gotten a dedicated computer and speakers for our BioSafety Lab 3 and I'm in music heaven. There's nothing like being in full PPE blasting music confident in the knowledge that you're completely alone and no one can hear you.

With that said, I present to you my BSL-3 playlist! I've handpicked these songs to provide a pick-me up when you most need it. Hopefully this will make the long hours fly by.

All the links I've posted will link to Spotify.

Enjoy!

Grimes – Genesis
Battles – Atlas
The Shins – Simple Song
M.I.A. – Bad Girls
Passion Pit – I'll Be Alright
Metric – Help I’m Alive (The Twelves Tabloid mix)
Sbtrkt – Wildfire (feat. Little Dragon)
Foster The People – Helena Beat
The Naked And Famous – Punching In A Dream
Electric Guest – This Head I Hold
Graffiti6 – Free
Hot Chip – Ready For The Floor
Passion Pit – Take A Walk
Peter Bjorn And John – Second Chance
The Naked And Famous – Young Blood
Passion Pit – Sleepyhead
Solange – Losing You
Kelis – Acapella
Crystal Castles VS Health – Crimewave
Die Antwoord – I Fink U Freeky
We Were Promised Jetpacks – Quiet Little Voices
Tegan And Sara – Closer
AWOLNATION – Kill Your Heroes
Sky Ferreira – Lost In My Bedroom
Sleigh Bells – Riot Rhythm
Cults – Abducted
Dum Dum Girls – Wrong Feels Right
Hot Chip – Motion Sickness

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!!

A momumentous undertaking

This is a space I hope to fill with my collected thoughts on life, literature and the pursuit of a Ph.D. After 8 years of studying science, after hours upon hours of research work in different labs, different states and soon to be different countries I only now have an inkling of what science and the real "pleasure of finding things out" is all about. Curiosity is not a subject taught in the American school curricula and I've managed to maintain mine intact despite all those years of clueless wandering. Hard work and diligence substituted for a real understanding of what the process of discovery encompassed. I'm slowly making my way through the dark forests of ignorance and this blog will hopefully detail that journey.