density gradient cell separation method
density gradient cell separation method
This experiment is mainly used to isolate cells.
Operation method
density gradient cell separation method
Principle
Density gradient solution was prepared, cells were centrifuged through the density gradient solution, and each gradient component was collected, diluted with culture medium, and cells were cultured.
Materials and Instruments
Cell samples Move Density gradient solutions were prepared by one of the following methods: Caveat 1. make sure that the trypsin digested cells form a single cell suspension. 2. Strictly follow the procedure and do not artificially brake the end of centrifugation. For more product details, please visit Aladdin Scientific website.
D-PBS 0.25% trypsin
Growth medium Growth medium + 20% Percoll 25 ml centrifuge tube Syringe or gradient collector 24-well culture plate Refractometer or densitometer Low-speed centrifuge
(a) Layered spreading method:
(i) Adjust the density of Percoll to 1.10 g/CC with an osmotic pressure of 290 mOsm/kg.
(ii) Mix Penroll with different ratios of regular medium to achieve the desired density range (e.g., 1.020-1.100 g/ml), typically 10 or 20 levels.
(iii) In a 25 ml centrifuge tube, using a pipette, syringe, and peristaltic pump, take 1 ml or 2 ml of each density and lay one density on top of the other, layer by layer, starting at the highest density to create a stepped density gradient. Alternatively, you can start from the lower density and use a syringe or peristaltic pump to inject the solution of the higher density to the lower level of the previous one. The latter method may be preferable.
The density gradient solution can be used immediately or left overnight.
(b) Use of gradient formers:
A continuous linear gradient can be achieved by mixing different concentrations of Percoll in a gradient formers. For example, mix 1.020 g/ml and 1.080 g/cc of Percoll (Fisons, Pharmacia, Buchler) in a gradient generator. 
(c) Centrifugation:
(i) Add medium containing Percoll at a density of 1.085 g/cc to a centrifuge tube.
(ii) Centrifuge for 1 h at 20,000 g.
(iii) Centrifugation produces an S-shaped density gradient, the shape of which is determined by the initial concentration of Percoll, the centrifugation time, the centrifugation force, the shape of the centrifuge tube, and the method of centrifugation. 
1. Trypsin-digested cells are re-suspended in medium supplemented with serum or trypsin inhibitor, making sure that it is a single-cell suspension.
2. 2. 2x107 cells are removed from the culture medium using a syringe, pipette, or a pipette with a thin end. 2 ml of cell suspension containing 2x107 cells is carefully flattened onto the top of the gradient.
3. Centrifuge the tubes upright on the benchtop for 4 h and allow the cells to settle freely at 1 g or centrifuge for 20 min at 100-1000 g. If the latter method is used, gradually increase the speed of the centrifugation at the beginning of the centrifugation and do not artificially stop the centrifugation near the end of the centrifugation.
4. Use a syringe or gradient collector (Fischler) to collect the cells from the cells and then centrifuge the cells into the culture medium. Collect the cells using a syringe or gradient collector (Fisons). 1 ml can be collected into a 24-well plate, while 0.1 ml can be collected on a microtitre plate. Samples should be taken at regular intervals for cell counting and density (β) determination of the gradient. Density measurements can be made with a refractometer (Hilger) or a densitometer (Paar).
5. Add an equal volume of medium to each well and mix well to ensure that the cells can touch the bottom of the well. After 24-48 h of incubation, change the medium to remove Percoll. 