Which gauge for proper shear force on certain cell dimension - (Jan/08/2014 )
basically I wish to know if there is a way to determine the proper needle gauge I have to use for lysis of adult rat cardiomyocytes.
My isolated cells are about 100-150 µm long. I have searched through many cell fractionation protocols, but I failed to find something like a rule of thumb for which gauge to use for which cell dimension.
The lysis (in hypotonic buffer) is supposed to be very gentle so to only target the outer cell membrane. Intact mitochondria and nuclei are crucial.
So does anyone know of a way? Or has experience with this? Or will I have to intricately establish this? A good starting point would be half the battle..
Thanks in advance!
If you need intact nuclei, I would recommend using Dounce homogenizers - like these ones, as they come with two different gauges to shear off the cytoplasmic membranes but leave the nuclei intact. Mitochondria will also remain intact.
Using a 21 gauge needle may also work, but you will still get a lot of broken nuclei.