PriCells-mouse mononuclear cell separation step-gradient removal of dead cells
Living cells (less dense) and dead cells (higher density) have different densities, which help to separate living lymphocytes from red blood cells.
High density solution: Ficoll-Paque (Ficoll and sodium diaphan; Pharmacia LKB) or Lympholyte-M (Cedarlane)
12ml or 50ml polypropylene centrifuge tube (better than polystyrene centrifuge tube)
Note: The density of Ficoll-Paque and Lympholyte-M is temperature dependent; the high density solutions used in this process as well as other commercial products are suitable for use at room temperature. Therefore, care should be taken to control the cell suspension, centrifugation and high density solution at room temperature. Otherwise, living cells will be lost at the density interface due to the sinking of living cells into the bottom of the centrifuge tube.
1. Suspend the cells with RPMI-5 complete medium and dispense into the centrifuge tube to make the number of cells reach 0.5Ã—10 8 ï½ž1Ã—10 8 cells/2ml (12ml centrifuge tube) or 1Ã—10 8 ï½ž5Ã— 10 8 cells / 5 ml (50 ml centrifuge tube).
2. Pipette 3 ml (when using a 12 ml centrifuge tube) or 5 ml (when using a 50 ml centrifuge tube) high-density solution, extend the tip to the bottom of the centrifuge tube, and slowly add the high-density solution to the bottom of the cell suspension.
3. Centrifuge at 800g for 15min at room temperature. In order to achieve good separation, it is best to turn off the "brake" switch of the centrifuge.
4. Using a 5 ml pipette, slowly move the pipette tip along the surface of the high-density layer, inhale the living cells floating above the high-density layer, and collect as low a density solution as possible. Move the living cells into another tube.
5. Add RPMI-5 complete medium (add 10ml to a small amount of cells, add 40ml when the amount of cells is large), and centrifuge at 200g for 10min at room temperature. repeat.
6. Suspend the cells in an appropriate amount of medium for the next step.
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