Link to abstract list.
On my way to Translational Science 2015 in Washinton DC
See you in a few days Union Station.
Cell Loss Through CD45 Depletion Process
Thursday Dec. 11, 2014: Performed a CD45 depletion on two EDTA tubes of 7.5 mL whole blood. Tube 1 straight depletion, tube 2 WB spiked with estimated 1000 MCF7 cells. 50 uL of concentrated 650 uL volume of WBC fraction isolated from buffy coat before depletion stored as reference. CD45 depletion performed on remaining faction. Post depletion fractions fixed with 4% formaldehyde on ice for 15 minutes(4 tubes total). (more procedures to come)
Today: Performed flow cytometry to sort for DNA+ cells, i.e. has nucleus and is not debris. Permebilized cells with 50 uL of 10x BD perm/wash into 450 uL volume and added 50 uL 10 ug/mL DAPI for staining nucleus.
Calibrated DNA+ with 1 reference sample. 1 depleted had 1350 nucleated cells. 2 depleted(spiked with 1000 MC7) had no cells. I must have lost the cells in the wash process from the fixation. Everyone says all washes have loss. Karen suggested to use higher RPM wash. Go to 2000 RPM on beckman. She points out I don’t care about damage to cells that already fixed.
My though is I need to develop some technology that allows me to do cell washes without loss. I have some ideas on how I can use the track etched polycarbonate membranes to do this. More to come.
My new blog ctchunter1.gregfutia.com
The purpose of this blog is to document and report my ongoing activities in developing technology for the identification and isolation of circulating tumor cells (CTCs).
First, CTCs are rare cells (1-50 per 10^6 white blood cells (WBCS) or 10^9 Blood Cells) shed from a tumor into the blood stream. The difficulty in identifying, isolating them and developing technology to do this, is due to their rare nature. The rarity is sometimes related through the analogy of identifying terrorists 1-50 in 10^9 of the world population and I think this analogy gives a perspective on the difficulties of the problem. This technology development is also in the scope of a PhD in Bioengineering at the University of Colorado Denver | Anschutz Medical Campus.
I setup this blog to detail, in public and open fashion, my current activities in engineering this technology. I’ve come to realize that I’ve collected a good amount of data on things that work and things that don’t and I want to disclose this findings and the data. Also, I’m hopeful that by disclosing them I can get feedback from you and develop them faster. This disclosure process is a delicate process since I’m also interested in publishing and developing IP on this work.
This is my second attempt at trying to setup a blog. First time failed due to a lack of commitment. I also wasn’t happy when blogger.com discontinued the ability to update to ones self owned server (in my case gregfutia.com) early on in the process because I don’t like losing control of my content to whoever is hosting.
CTCHunter1 was the name of the twitter handle I setup after I attended the 21st Cytometry Development Workshop at the end of October, 2014. It was a great conference and afterwards I realized I need to use the internet better to say in touch.
I set this website up using wordpress.org. It was pretty easy after downloading the tar.gz and setting up the databases. The initial setup time really was ~5 minutes. So far I’ve been pretty happy with it.