Product Name ：Human peripheral blood lymphocyte isolation solution

Product Specifications ：200ml

Storage conditions::RT， light-proof

Validity period:12 months

Product Introduction:This product is a sterile, low endotoxin level density gradient separation solution used for separating human peripheral blood lymphocytes. The separation principle is based on the density difference of blood cells (red blood cell and granulocyte density is1.090 g/mLabout; The density of lymphocytes and monocytes is1.075~1.090 g/mLPlatelets are1.030~1.035 g/mL）By centrifugation, cells of a certain density are distributed according to a corresponding density gradient, thereby separating lymphocytes from human peripheral blood or umbilical cord blood.

Product metrics:density1.077±0.001g/mLosmotic pressure290~350 mOsm/kg H2Osterile0.1Mmmembrane filtration

Storage conditions:This product is sensitive to light and should be stored at room temperature away from light. Shelf life1 Year. After sterile opening, store at room temperature.

Operation steps:1. Take fresh anticoagulated whole blood（EDTASodium citrate or heparin anticoagulants can be used) or fibrinogen free blood, diluted with equal volumes of whole blood and tissuePBSDilute whole blood.2. Add an appropriate amount of separation solution to the centrifuge tube (when the diluted blood volume is less than3mLWhen, join in3mLSeparation liquid; greater than or equal to3mLAdd an equal volume of separation solution. But the total volume of the two should not exceed two-thirds of the centrifuge tube, otherwise it will affect the separation effect. Spread the diluted blood evenly above the separation liquid surface, paying attention to keeping the interface between the two liquid surfaces clear. (You can use a Pasteur pipette to draw blood, and then carefully spread the blood flat on the separation solution, as the density difference between the two will form a clear layered interface. If there are many samples and the sample addition time is long, it is normal for red blood cells to clump and sink before centrifugation.)

3. Room temperature, horizontal rotor500~1000g, centrifugation20~30minThe larger the volume of blood, the greater the centrifugal force requiredThe longer the time, the better the separation conditions need to be explored, and the maximum centrifugal speed should not exceed1200g）.4. After centrifugation, there will be obvious stratification: the top layer is the diluted plasma layer, and the middle layer is the transparent separation liquid layer. The plasma and separationThe white membrane layer between the centrifuges is the lymphocyte layer, while the bottom of the centrifuge tube contains red blood cells and granulocytes.5. Carefully extract white membrane cells into15mLIn a clean centrifuge tube,10mL PBSOr wash the white membrane cells with cell washing solution.250g, centrifugation10min.6. Discard the supernatant,5mLofPBSOr resuspend cells in cell cleaning solution,250g, centrifugation10min.7. Repeat the steps68. Discard the supernatant and resuspend the cells for future use.

Separation purity:The purity of lymphocyte separation using human lymphocyte separation solution is greater than90%.

Notes:A.Before opening, invert and mix thoroughly. This separation solution is a sterile product. To extend the storage time of the separation solution, please open it under sterile conditions to avoid microbial contamination..B.The separation liquid should always be kept at room temperature when used（18℃~25If the indoor temperature is low, the separation liquid can be preheated.4Centrifugation at ℃ or lower temperatures may lead to increased contamination of red blood cells in the white membrane layer.C.It is best to use fresh anticoagulant blood samples (blood collection)2 hTo maintain the activity of lymphocytes, freezing and refrigeration should be avoided.D.Dilute blood or wash cells, do not use containingCaTheMgIonic buffer and culture medium, whose components can cause blood cell agglutination, greatly reduce cell yield and purity.E.Some plastic products, such as polystyrene, may cause cell wall adhesion due to their electrostatic effects, which can affect the separation efficiency.F.The viscosity or temperature difference of blood samples may affect the separation effect. The centrifugation speed and time can be adjusted to explore the optimal separation conditions.G.Excessive absorption of lymphocyte layers and separation fluid layers can lead to the extraction of granulocytes at the boundary of the separation fluid, resulting in an increase in the number of mixed granulocytes; Excessive absorption of plasma layer may lead to contamination of plasma proteins and platelets in lymphocytes.H.If further cultivation of isolated cells is required, attention should be paid to aseptic procedures during blood collection and separation to avoid microbial contamination.I.The cell dispersion coefficient and cell charge of different animal blood in different specific gravity separation solutions are different. When formulating the separation solution, users should provide the required specific gravity of the separation solution, animal species, and the name of the separated cells.