Percoll™ consists of silica particles (15-30 nm diameter) coated with non-dialyzable polyvinylpyrrolidone (PVP). Free PVP is present at only 1-2%. Percoll™ is non-toxic, almost chemically inert and does not adhere to membranes. Percoll™ gradients can be formed within the density range of 1.0-1.3 g/ml, and are iso-osmotic throughout.
1) Dilute stock Percoll™ in HBSS (Invitrogen) to 280-320 mOsm/kg H2O and 1.070 g/ml density. Various dilutions o stock can be made to lower densities by using the formula:
Vy=VI x (pI-p) (p-py)
Vy= volume density
VI= volume stock Percoll™
pI=density stock Percoll™
py= density diluting medium
p=desired final density
2) Pour 20 ml of 1.070 g/ml Percoll™ into a 25 ml ultra-centrifuge polycarbonate tube.
3) Spin the tube at 30,000 g for 20 minutes at 4 C using a fixed angle ultra-centrifuge rotor such as Beckman 30. Do not use break and use the "decelerate" option on ultra-centrifuge.
4) Layer up to 1 x 10E8 mononuclear cells diluted in HBSS on top of the gradient.
5) Spin the gradient at 400 g for 30 minutes at 4 C. Do not use brake.
6) Separate the resulting cellular bands using a Pasteur pipette or syringe.
7) Wash the cells at least 5 times using 10 ml of HBSS per wash.
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