Immune cell isolation techniques are developing in the direction of more and more "fine", immune cell subpopulation and even micro-immune cell isolation and identification techniques have also matured and have been applied in the laboratory, such as through the immunomagnetic beads can be obtained a large number of highly purified immune cell subpopulations of various immune cell subpopulations, and through the flow cytometer can be sorted to the immune cell populations of the expression of specific antigens. Of course, some conventional immune cell isolation methods, such as separation of peripheral blood single nucleated cells by density gradient centrifugation, are also very simple and practical, and are still widely used, but are now more often used for preparation and cell enrichment prior to immunomagnetic bead sorting or flow cytometry sorting. In conclusion, there are many immune cell separation techniques to choose from, but it is important to select the most practical immune cell separation method according to the characteristics of the immune cells needed for the study, the requirements of the study content and the conditions of the laboratory.
Principle
The basic principle of peripheral blood single nucleated cell separation is based on its cell density (1.075 ~ 1.090) and other cells (erythrocytes and neutrophils density of 1.090, platelets 1.030 ~ 1.035) is different, using the density of 1.075 ~ 1.092 between the near-isotonic solution (layered solution) for density gradient centrifugation, so that the different densities of cells according to the corresponding density gradient distribution, thus separating the single nucleated cells. The cells are distributed according to the corresponding density gradient, so that the single nucleated cells can be separated.
Operation method
Isolation of peripheral blood single nucleated cells
Principle
The basic principle of peripheral blood single nucleated cell separation is based on its cell density (1.075 ~ 1.090) and other cells (erythrocytes and neutrophils density of 1.090, platelets 1.030 ~ 1.035) is different, using the density of 1.075 ~ 1.092 between the near-isotonic solution (layered solution) for density gradient centrifugation, so that the different densities of cells according to the corresponding density gradient distribution, thus separating the single nucleated cells. The cells are distributed according to the corresponding density gradient, so that the single nucleated cells can be separated.
Materials and Instruments
Reagents: Move The basic procedure for isolation of single nucleated cells from peripheral blood can be divided into the following steps: Caveat 1. Generally, 1 x 10 per milliliter of healthy adult blood can be isolated.6～2 x 106Single nucleated cells. The viable cells isolated by this method should be more than 95%. 2. In Procedure 2, isovolumetric dilution of the blood reduces red blood cell coagulation and improves lymphocyte harvest; this step may be omitted. 3. In order to maintain the activity of lymphocytes, the cells should be separated as soon as possible after blood collection. 4. This procedure is also applicable to the separation of single nucleated cells in tissues. For blood that has not been anticoagulated, streptokinase can be used to dissolve the clot first, and then separated. 5. For small animals (e.g. mice), blood can be taken from the eye socket or from the severed head, anticoagulated, and the blood diluted 1:1 with PBS to increase recovery. 6. Many studies require the application of single nucleated cells from mouse spleen, thymus, and lymph nodes. For these cells, the conventional method is to prepare a single-cell suspension of mouse spleen, thymus, and lymph nodes, and then lysing the erythrocytes in it with erythrocyte lysing solution, and then washing and discarding the lysate and centrifugation of the cells obtained by Ficoll-Hypaque stratification in a density-gradient manner to remove the cells and the remaining erythrocytes. The cells and remaining erythrocytes are removed by density gradient centrifugation in Ficoll-Hypaque lysate. Different sources of mouse mononuclear cells can be prepared. For more product details, please visit Aladdin Scientific website.
1. Sterile heparin solution
Heparin anticoagulant solution containing 125~250 IU/ml was prepared in sterile saline and stored at 4 ℃.
2. Lymphocyte stratification solution (Ficoll-Hypaque, density 1.077 g/L ± 0.001 g/L). 3.
3. Hanks balanced solution or saline or PBS, pH 7.2-7.4, stored at 4 ℃ and protected from light.
4. RPMI 1640, complete culture medium containing 10% fetal bovine serum. 5. 15 ml or 50 ml of RPMI 1640.
5. 15 ml or 50 ml polypropylene conical centrifuge tube. 6.
6. Trypanosoma cruzi blue staining solution.
Instruments:
Low-temperature centrifuge, sterile dropper, pipette, blood cell counter, inverted microscope and ultra-clean table.
1. Collect fresh blood aseptically and anticoagulate it with heparin (0.1 ml of heparin solution per ml of blood). If the volume of blood is large, centrifuge the blood at 2000 r/min or 400 g for 20 minutes at room temperature to remove the upper plasma layer and the leukocytes at the erythrocyte junction.
2. Dilute the whole blood or leukocyte-rich blood with an equal volume of PBS or Hanks. 3. Dilute whole blood or leukocyte-rich blood with an equal volume of PBS or Hanks solution.
3. Add an equal volume of lymphocyte fraction to a 15 ml conical centrifuge tube and bring to room temperature (18-25°C).
4. Tilt the tube at an angle of 45° and draw up the diluted blood sample with a glass pipette, spreading it slowly along the wall of the tube 1 cm above the surface of the fractionation solution and on to the top of the lymphocyte isolate, being careful not to disturb the interface of the liquid layers.
5. After leveling, place the sample in a horizontal centrifuge and centrifuge the sample in a horizontal position at room temperature (optimally 18-20°C). Centrifuge at room temperature (optimal temperature is 18-20 ℃), 2000 r/min or 400 g for 20 minutes, blood stored for more than 2 hours should be centrifuged for 30 minutes.
6. Remove the tube from the centrifuge smoothly, after centrifugation, the bottom layer is the red blood cells and granulocytes, the intermediate layer is the stratified fluid, and the top layer is the plasma and the diluent. At the junction of the plasma layer and the stratified fluid is a cloudy grayish-white layer rich in individual nucleated cells (including lymphocytes and individual nucleated cells).
7. Insert a capillary pipette gently into the layer of individual nucleated cells, and suck up the layer along the wall of the tube and put it into another tube that has already been pre-filled with 10 ml of physiological saline or Hanks' liquid or RPMI 1640 complete culture medium. Alternatively, the upper layer of plasma and the diluent can be discarded, and the layer of cells can be pipetted into another tube.
8. Centrifuge the obtained diluted single nucleated cells at room temperature, 1500 r/min or 250 g for 10 minutes, and discard the supernatant. Repeat the washing process one or two times to remove platelets, separating medium and anticoagulant material.
9. After checking the viability of the cells by Trypanosoma cruzi blue staining, the cells are counted and diluted with PBS or suitable medium as required.