Equal density centrifugation to remove red blood cells and dead cells
Equipment and reagents:
All supplies and reagents that come into direct contact with the cells must be sterile.
1. A test tube with a volumetric scale (conical bottom);
2. Pasteur pipettes (short and long);
3. 10ml graduated straw and ear wash or pipetting device;
4. Benchtop centrifuge;
5. Lymphocyte separation solution or similar reagent;
6. Medium;
experimental method:
1. Resuspend the cells to a high concentration of 5 x 10 6 - 10 7 / ml. 10 ml of lymphocyte separation solution was added to a centrifuge tube, and 5 ml of the primary cell suspension was carefully added to the lymphocyte separation surface using a Pasteur pipette or a 10 ml pipette. The container is tilted at an angle and the cells are slowly dropped down the wall of the container instead of directly onto the surface of the lymphocyte separation. The goal is to obtain a clear interface between the two liquids for better separation;
2. Do not brake the 1000r/min centrifuge tube for 20min (Note: slow deceleration during centrifugation can form a clear interface between the lymphocyte separation solution and the medium);
3. Carefully remove the container from the centrifuge and observe the interface between the two liquid surfaces to reveal a milky yellow cell band. Red blood cells and dead cells form a sediment that sinks to the bottom of the container;
4. Using a Pasteur pipette to aspirate the cell layer visible on the interface and transfer to a centrifuge tube;
5. Add fresh medium to the collected cells and mix. Centrifuge at 1000r/min for 5min, repeat twice, wash away the lymphocyte separation solution on the cell surface;
6. Resuspend the cells in a known volume of medium for cell counting and viability assays;
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