Materials and reagents 1. Heparin and other anticoagulant 2.3% gelatin solution 3g gelatin, dissolved in 0.9% sterile saline 100ml, sterilized at 114.3 ° C for 10min, cooled and stored in a refrigerator at 4 ° C. When used, preheat at 37 ° C for 10 min. 3. Ash liquid (two) method of operation 1. Blood collection and anticoagulation, that is, red blood cells. Because the ratio of red blood cells to white blood cells is very different, white blood cells are usually mixed in, negligible. 3. If you need to store and reserve, use A's solution as an anticoagulant to collect blood, mix it and store it in a refrigerator at 4 °C for 2 weeks. The activity is acceptable. Lymphocyte separation technique (1) Materials and reagents 1. There are two types of lymphocyte stratification: (1) Polysucrose - diatrizoate solution: polysucrose solution, trade name Ficoll, molecular weight 400,000, more formulated into 40 ± 1% (W / V) aqueous solution, also available in dry powder. When applied, 9% solution was prepared with distilled water. Meglumini diatrizoici, trade name Vrografin, structural formula 3,5-diacetylamino 2,4,6 triiodobenzoic acid 1-deoxy-methylaminosorbitol. The content is 60% or 75%, and the volume of each ampule is 20ml. It is often used for human organ angiography. When applied, take 60% of 60% diatrizoate stock solution and add 15.38 ml of double distilled water, which is 33.9% diatrizoate. Preparation of 1.077±0.001 polysucrose-diatrizoate: 9% polysucrose solution 24 parts 33.9% diatrizoate solution 10 parts can be mixed. When necessary, the specific gravity can be measured. Need to be spare, can be filtered by G5 funnel or autoclaved at 114.3 °C for 15min, stored at 4 °C. Generally can be saved in March. To prepare different proportions of stratified liquid, it can be calculated according to the following formula: where dm is the specific gravity of the stratified liquid, d1 and d2 are the specific gravity of 9% polysucrose and 33.9% diatrizoate, respectively, V1 and V2 are Their volume. If a layered solution of 1.077 specific gravity of sucrose-diatrizoate is to be prepared, the ratio of 9% of sucrose to 33.9% of diatrizoate should be 100..46.3. (2) diatrizoate-dextran solution (dextran) solution 34% diatrizoate solution 10 parts 60% dextran solution 12.5 parts mixed, which is a layered liquid with a specific gravity of 1.07 to 1.09. Dispense in brown bottles and store in a refrigerator at 4 °C. 2. 3% gelatin solution was weighed 3 g of gelatin, dissolved in 0.9% sterile physiological saline, the final volume was 100 ml, autoclaved at 114.3 ° C for 10 min, stored in a refrigerator at 4 ° C after cooling, and preheated at 37 ° C for 10 min. 3. Hank's solution. (2) Low speed centrifuge operation method 1. Take anticoagulation, natural sedimentation, such as the blood of the horse animal, can be directly erected on the test tube rack, let it naturally settle for 1h, take the upper layer of plasma. If the blood of cattle and sheep is very slow, add 3% gelatin solution, mix it and let it settle naturally. After 1h, take the upper layer of plasma. 5. Take 2 ml of cell stratified solution in a centrifuge tube, while using a capillary pipette to draw about 2 ml of plasma (upper layer of plasma that naturally settles for 1 h), gently add to the stratified solution, and directly add anticoagulation. 8. Wash with Hank's solution, centrifuge, and finally prepare the appropriate white blood cell concentration. Count as necessary. (three) matters needing attention 1. Be careful when adding plasma or blood to the stratified solution. Slowly do not disturb the liquid layer or shake it. It is also possible to add the stratified liquid to the upper layer of the plasma. More mini centrifuge information login: Dried Shiitake Mushroom,Dried Shiitake,Organic Dried Shiitake Mushrooms,Shiitake Mushroom Dehydrated Guangyun Agricultural Biotechnology (Jiangsu) Co., Ltd , https://www.7-mushrooms.com
2. Further processing can be done depending on the use of the red blood cells. If calculating red blood cells, you can directly dilute the meter. If you need to do a complement binding reaction, or other red blood cell reaction, you need to wash the red blood cells thoroughly to remove the plasma attached to the surface of the red blood cell membrane. It is usually washed continuously with an isotonic diluent, centrifuged at 2 000 r/min for 10 min, and repeated 3 to 4 times.
Centrifuge at 2.2 000 r/min for 10 min and discard the supernatant.
3. The pellet was suspended in Hank's solution and centrifuged at 2 000 r/min for 10 min.
4. Repeat step 3 once and then use the nutrient solution to make the white blood cells into a suspension. This fluid contains the entire white blood cell population, including granulocytes, lymphocytes, monocytes, and parts of red blood cells. Available for white blood cell counting.
6. Centrifuge at 20 000 r/min for 20 min in a horizontal rotor centrifuge. After centrifugation, it can be seen divided into multiple layers, the lowermost layer is red blood cells, the middle layer is stratified liquid, and the uppermost layer is plasma. Between the plasma layer and the stratified liquid is a thin layer of denser white layer, which is a single nuclear cell layer.
7. Insert into the mononuclear cell layer with a capillary pipette and pipette the layer into another tube.
2. It is very important to maintain the activity of lymphocytes, so in general, blood is collected and immediately separated.
3. The layer of lymphocytes separated by stratified fluid is actually a mononuclear cell layer, including monocytes, excluding granulocytes. To isolate pure lymphocytes, mononuclear cells are removed as follows, or monocytes are harvested.