From Laurent Brossay, Brown University
Liver Grinding
1. Harvest liver and put in media.
2. Use a 6-well tissue culture plate, a 70 um nylon cell strainer (VWR # 21008-952), and a 3cc
syringe plunger
(this end), and a frozen tissue cold plate to grind the liver.
3. Add 2-3 mL of PBS Serum to each well.
4. With liver inside of cell strainer grind liver using end of 3cc plunger until all cells have
passed through strainer. (Grind liver until PBS Serum is saturated with cells, transfer liquid
to centrifuge tube, then add another 2-3 mL PBS Serum to well through the strainer.
Continue to grind liver.
5. Once cells have been collected, remove and discard liver, syringe plunger, and cell strainer.
6. Wash well with 1 ml PBS Serum and collect remaining cells. Repeat this 2-3 times until all
cells are collected.
7. Centrifuge at ~ 1400 rpm for 10 minutes. Carefully remove and discard supernatant – cell
pellet may not be completely solid and may come out with supernatant – Wash cells 3 times
with PBS Serum and centrifuge at 1200 – 1300 rpm.
8. After last wash, resuspend cells in 8 mL room temperature 40% Percoll. Carefully pipet
this 8 mL onto 3 mL room temperature 70% Percoll being careful not to mix fractions.
Centrifuge at 2400 - 2500 rpm for 20-25 minutes at room temperature with NO BREAK.
9. Remove Fat Layer being careful not to break it into pieces. Collect the Ring. Wash the cells
at least 1 time before use.
Fat Layer
Ring
Red Blood Cell Pellet
From Laurent Brossay, Brown University