Lymphocyte Separation Medium (Rabbit)

This product is a gradient density separation solution suitable for the separation of rabbit peripheral blood lymphocytes. Its principle is mainly based on the density difference between different peripheral blood cells (the density of red blood cells and granulocytes is about 1.090 g/mL; Platelet is 1.030-1.035 g/mL; Lymphocyte and monocyte are 1.075-1.090 g/mL) and are separated from peripheral blood by gradient density centrifugation. This product is sterile, low endotoxin level ready-to-use separation solution with a density of 1.083 ± 0.001 g/mL (20℃). The product is optimized on the basis of traditional Ficoll-meglumine to maintain the stability of the separation solution for a longer time after blood addition, the operation is simple and the isolated lymphocytes are of high purity and good condition.

Ship with wet ice;Store 2-8℃ away from light, valid for 12 months.

For the separation of 1 mL of rabbit peripheral blood lymphocytes, the volume ratio of blood to lymphocyte separation medium is 1:1-1:2, with appropriate adjustments within this range; Care should be taken when selecting the centrifuge tube that the total volume of blood and lymphocyte separation medium does not exceed two-thirds of the volume of the tube.

1. Take 1 mL of fresh anticoagulant whole blood (heparin, EDTA, sodium citrate and other anticoagulants can be used), dilute with an equal volume of PBS (G4202 recommended) or Hanks buffer (recommended g4203) contains no calcium and magnesium to obtain 2 mL of diluted whole blood.

2. Pipette 3 mL of peripheral blood lymphocyte isolation solution from rats and mice into a 15 mL sterile centrifuge tube (EP-1500-J is recommended).

3. Tilt the centrifuge tube at 45° and slowly add 2 mL of diluted blood along the tube wall into the centrifuge tube, so that the blood is lies flat on the upper layer of the peripheral blood lymphocyte separation medium of rabbit.

4. It is recommended to use a horizontal head centrifuge, place the tube in the horizontal head adapter, reduce the speed of the centrifuge (3-5 speeds are appropriate) and centrifuge at 800 x g for 25 min at room temperature.

5. After centrifugation, the tube is gently held on a tube rack and a clear stratification is observed: the uppermost layer is the plasma layer; the upper middle layer is the lymphocyte layer; the lower middle layer is the lymphocyte separation medium layer; and the lowermost layer is the erythrocyte and granulocyte layer (refer to the attached figure).

6. Remove the uppermost plasma layer and carefully aspirate the tunica albuginea layer (lymphocyte layer) into a new sterile centrifuge tube.

7. Wash with 8 mL of PBS (recommended G4202) or other buffer, centrifuge at 100 x g for 10 min, then discard the supernatant

8. Repeat Step 7 (Optional);

9. Resuspension of lymphocytes in the required medium or buffer according to the purpose of the experiment.

10. The purity of the lymphocytes can be further improved by planting the resuspended lymphocytes into a culture dish or flask, incubating in the incubator for 1-2 h, and then transferring the absorbed cells to a new culture dish or flask (optional).

1. The product must be fully equilibrated and mixed upside down at room temperature before use. The proper temperature for separation is 18-25℃.

2. To maintain the activity of lymphocytes, it is best to select fresh anticoagulation blood within 2 hours of blood collection; If further culture and test of the isolated lymphocytes is required, please pay attention to the aseptic operation during blood collection and separation.

3. Diluting or washing the buffer, do not use the buffer contains calcium and magnesium ions to avoid blood cell aggregation

4. Excessive absorption of components outside the tunica albuginea layer of lymphocytes will cause some granulocytes or platelets at the junction to be mixed.

5. Differences between blood samples may have an impact on the separation results. The centrifugal force and time can be adjusted appropriately according to the actual situation. The reference centrifugal force and time range are 500-1000 g and 20-30 min.

6. It is normal for red blood cells to settle after mixing blood and lymphocyte separation medium for a certain time.

7. For your safety and health, please wear safety glasses, gloves, or protective clothing.

Attachment: Schematic diagram of each layer before and after separation

This product is only for scientific research purposes, not for clinical diagnosis!