Hi Diana,

There are a couple of strategies you may want to try, but could your clarify a few questions about your experiment?

1) Do you have a measure of cell viability after sorting? Do you include propidium iodide or a similar dye during your flow experiments to make sure you're recovering live cells?

2) Are these cells derived from tissue culture or from tissues, and if tissues, fresh or frozen? Related to question 1, are these cells fixed?

3) Did you try adding glycogen during the DNA precipitation step?

Thanks,

Barry

Barry Zee thanks for your attention! About your questions:

1) We couldn't measure cell viability because we used intracellular staining with permeabilization. Cell viability in suspensions (trypan blue) was about 70% before cell staining.

2) These cells were derived from frozen arteries using enzymatic disaggregation;

3) I have never added glycogen or dextrans but I'm going to.

Do you think the lack of cell viability assay is critical for study results?

I'm looking forward to your reply!

Sincerely,

Diana

Hi Diana,

Hmm, so there's approximately 30% dead cells before permeabilization, and once you permeabilize and incubate with your antibody, I assume you won't be able to distinguish the previously live (now dead) cells and previously dead cells? So when you sort 3000 fixed cells, a sizeable fraction of those cells were originally dead? The dead cells may contain fragmented/degraded genomic DNA that can reduce the apparent efficiency of DNA isolation later on (the larger and more intact the DNA is, the easier it is to precipitate/bind to column).

I think increasing your initial cell viability percentage prior to permeabilization may improve your results, perhaps using an initial sort for live cells and then immediately permeabilize for your antibody incubation or improving your enzymatic disaggregation method. There are also magnetic-bead based approaches to depleting dead cells if that's easier for your experiments. What is your method of fixation?

Incubating with glycogen for a sufficiently long period is probably the easiest thing to implement first to try to boost your DNA yield. There are also DNA low-binding tubes commercially available that may help improve recovery of DNA. Finally 3,000-100,000 sorted cells is a large range, so I assume you are already adjusting the volume of phenol/chloroform appropriately depending on the actual sorted number?

Thanks,

Barry