An insert for a centrifuge tube suitable for use in density gradient separation is described. The insert includes a member sized to fit within the tube for dividing the tube into a top portion and a bottom portion. Optionally the insert has a support extending or depending from the member for positioning the member within the tube. At least two openings are located on the member so that a first opening is closer to a bottom end of the tube relative to a second opening when the insert is positioned in the centrifuge tube. Also described are methods for separating a target population of cells from a sample using the insert for a centrifuge tube.