Quantifying Cell Lysis or the Presence of DNA - (Nov/17/2008 )
I am a Mechanical Engineering PhD student working on a microfluidics device to be used to perform DNA analysis. Part of this project includes lysing cells in a sample. We are considering several different methods of lysis that might work well on our small scale. In this project we would want to lyse bacteria and mammalian cells.
The problem I am having is with determining whether or not the cells have been lysed, and quantifying what percent has been lysed. We have a Vanguard 1486 FL fluorescent microscope (http://veegee.thomasnet.com/item/fluorescence-microscopes/ence-microscopes-professional-level-1400fli-series/1486fl?&seo=110) in which at 100x objective magnification, I can clearly see the red blood cells which we are attempting to lyse. However it is hard to tell even if the cells have been lysed. In the case of the red blood cells I was simply trying to see if they had been lysed, not extract DNA.
What might be some easy and relatively cost effective ways of determining cell lysis? Would there be a way to use some particular dye or fluorescence? In the end our goal is to extract the DNA from the cells to be analyzed in the device. So at this point I would like some way to quantify either if the cells have been lysed, or if DNA is present in the sample.
Any advice you could give would be greatly appreciated.
The problem will be that unless you have a intact sample for comparison to your lysed, you will have difficulty quantifying how much has been lysed. There are a range of dyes that can tell you if cells are alive or dead, and there will be things that can identify cell debris, but complete lysis of a cell means that all the components are in solution and hence hard to detect.
Your best bet will be to run a control sample next to your lysed and do a count of cell numbers on each. FACS is a good way of doing this and is essentially a fluorescent microscope with a camera that identifies each time a cell labelled with a fluuorescent dye passes the sensor.
There is a paper, which talks about development of lysis procedures on a chip. I am not sure if this particular paper relates to your experimental set up, but it might give you an idea and comparison.
Evaluation of cell lysis procedures and use of a micro fluidic system for an automated DNA-based cell identification in interplanetary missions
J.A. Hall, E. Felnagle, M. Fries, S. Spearing, L. Monaco and A Steele
Planetary and Space Science
Volume 54, Issue 15, December 2006, Pages 1600-1611
Lysing cells and releasing DNA from cells are not necessary the same thing. For RBC lysis you could measure the Hb in the supernatant after spin down the ghosts. However, if you goal is to extract DNA later, RBC lysis may not be the right model. Your lysis solution might cause the release of Hb but not DNA. For DNA extraction you need to completely disrupt the cell membranes. However, lysing different cells to extract DNA is well estabolished, why would you try to re-invent the wheel? Is existing lysis solutions imcompatible with your device?