CyTOForum

Hi everyone,

I am actually working on the optimization of a protocol for brain single cell suspension for CyTOF.

In the last few months I had to change 4 PSI due to clogging. The company thinks they are caused by the nature of my samples ( I had the same problem with PBMC) and for the next experiment they suggested me to try to use benzonase (https://www.sigmaaldrich.com/US/en/prod ... dlEALw_wcB)

I never used this reagent and I can't find any protocol about how to prepare it. Second, in which part of the staining should I use it? During the homogenization part or during the staining part?

Thank you in advance for your help

Hi Elisa,

Benzonase is a nuclease. It's added to media to help chew up any DNA released from dead or dying cells (cell processing, freeze-thaw, etc).

We add it at 1/10,000 to complete RPMI+FBS+Pen-strep when we're thawing cryoPBMCs. So, 1uL/10mL of media. I usually warm my media first, then add it just before I pull the cells out of the LN2. I then thaw each sample and add it to the Benz-media, spin down, resuspend in more Benz-media, and spin down again before counting cells and going on with my experiment.


Are you homogenizing and then going directly into staining, or are you homogenizing and then freezing?


Mike