胰酶 配制（Pancreatin preparation）

胰酶 配制（Pancreatin preparation） 胰酶 配制(Pancreatin preparation) I. equipment and reagents: Dry powder type culture medium, trypsin, penicillin, streptomycin, purified water system, electronic balance, pH meter, magnetic stirrer. Concrete steps: (1) preparation of water: The water used for cell culture must be very pure, free of ions and other impurities. Need to use double distilled water, three fresh distilled water or pure water. (2) preparation and disinfection of PBS (also used in other BSS, such as Hanks, D-Hanks liquid preparation): Volume 1, dissolved: drug (NaCl 8.0g, KCl 0.2g, Na2 HPO4 H2 1.56g KH2 PO4? O, 0.2g) with double distilled water poured into the beaker, glass rod to stir, then fully dissolved, pour the solution into a volumetric flask of accurate volume to 1000ml, it is PBS newly prepared solution. 2, in the solution bottle to be sterilized: PBS into the solution bottle (bottle drip large), covers the rubber cap, and inserted the needle into the pressure cooker 8 pounds of disinfection for 20 minutes. Attention should be paid to the use of distilled water to supplement evaporated water after autoclaving. (3) preparation and disinfection of trypsin solution: Trypsin acts as a means of hydrolyzing proteins between cells, thereby allowing cells to disperse. Different tissues or cells react differently to pancreatic enzymes. The activity of pancreatin dispersed cells was related to its concentration, temperature and time. When the pH was 8 and the temperature was 37 DEG C, the function of pancreatin was the strongest. When using pancreatin, should grasp the concentration, temperature and time, so as not to cause excessive cell damage caused by digestion. Because Ca2+, Mg2+ and serum and protein can reduce the activity of pancreatin, so the preparation of pancreatin solution should be chosen without Ca2+, Mg2+, BSS, such as: D-Hanks solution. When terminating digestion, the effect of trypsin on the cell can be terminated with serum cultures or trypsin inhibitors. 1, the trypsin by trypsin concentration was 0.25%, with the electronic scales accurately take double distilled water soluble small beaker (if the distilled water need to adjust the PH to about 7.2) or PBS (D-hanks) solution. Stir and mix overnight for 4 hours. 2, injection filter filtration disinfection: trypsin with a good solution to filter for injection in a super clean bench (0.22 micron membrane filtration sterilization). They are then packed in small bottles and stored at 20 degrees for use. (4) preparation and disinfection of green and Streptomycin Solution 1, the use of pure water (distilled water) to 15 pounds of high pressure sterilization for 20 minutes. 2, the specific operations are completed in the ultra clean taiwan. Penicillin is 800 thousand units per bottle, with a syringe with sterile double distilled water 4ml. Streptomycin was 1 million units / bottle, add 5ml distilled water, each 200 thousand units per milliliter. 3, the use of dissolved into the culture medium, so that the concentration of streptomycin in the final 100 units /ml. 1 units = 1 micrograms (5) preparation and disinfection of RPMI1640: 1, pH, dissolved, constant volume: first with the medium volume 2/3 double distilled water based powder and cultured with double distilled water washing bag 2-3 (washing liquid added medium), stir to dissolve powder, and in accordance with the packing instructions added to certain drugs. Then, the 0.5ml was added to the medium by syringe, and the concentration of streptomycin was 100 units at last, /ml. Then adjust PH to about 7.2 with an equivalent hydrochloric acid and NaOH. Finally, allow it to 1000ml and shake it well. 2 、 install the filter: when installing, first install the bracket, put the filter membrane according to the regulation, and connect the stainless steel filter and bracket with screw. Then remove the support legs are good to be disinfected with cloth. 3, filtration: usually filter filtration medium good preparation. The filter is usually filtered in a super clean bench. 4, packing: the filter will be divided into small bottles of culture medium, built in 4 degrees refrigerator, stand-by. 5. Add 1ml glutamine solution to the 100ml medium before use (two weeks at 4 DEG C). (6) inactivation of serum: Calf serum is commonly used in cell culture, Serum bought in 56 DEG C water bath inactivated after 30 minutes, then after filtration can be added to the medium in use. (7) HEPES solution: HEPES chemical name a HEPES (N '-a-hydroxythylpiperazine-N' - ethanesulfanic acid). Non-toxic to cells. It is a hydrogen ion buffer that can control the constant pH range for a long time. Using the final concentration of 10-50mmol/L, 20mmol/LHEPES contained in the general culture buffer capacity can be achieved. 1mol/L HEPE buffers are prepared as follows: Weigh accurately HEPTS 238.3g, adding three fresh distilled water volume to 1L. The filter is sterilized and stored at 4 DEG C after partial packing. Note: because the HEPES is now about 10g package of small bottles, it can be flexibly prepared according to the actual situation, but to ensure that the ultimate concentration of HEPES in the culture medium is still 20mmol/L. Such as: take 4.766 grams HEPES dissolved in 20ml three steamed water, filter sterilization, can completely (20ml) into 1L culture medium, or each 100ml culture medium can add 2ml. (8) glutamine: The synthetic medium contains large amounts of glutamine, and its function is very important. Cells need glutamine to synthesize nucleic acids and proteins, and glutamine deficiency leads to cell growth failure and even death. A certain amount of glutamine should be added in the preparation of various cultures. Because glutamine is unstable in solution, it can be broken down for 1 weeks at 4 DEG C, so it should be prepared separately and stored in a -20 temperature refrigerator, and the culture medium is added before use in 50% minutes. The glutamine solution should be added to the original glutamine when stored in the refrigerator for 2 weeks or more for more than 4 weeks. The content of glutamine in normal culture medium is 1 ~ 4mmol/L. 200mmol/L glutamine solution can be prepared for storage and added with culture medium. The preparation method for glutamine 2.922g dissolved in three water added to 100ml into 200mmol/L solution, stirring after dissolution, filtration, vials, -20 C preservation, can use 100ml to add 1ml glutamine in the solution culture. (9) preparation of heparin solution: Heparin containing culture solution can increase the purity of endothelial cells, and the final concentration of heparin in whole culture solution is 50ug/ml. Because the market is mostly heparin sodium, packaging is about 0.56 grams / bottle, when it is formulated, it can be dissolved in 100ml three steamed water, fixed volume overnight, and then filter sterilization, small bottles, storage temperature of - C. When in use, add 1ml to the 100ml medium (as accurately as 0.9ml). (10) type I collagenase: 0.1% type I collagenase solution is prepared and sterilized like trypsin. Note: since type I collagenase molecules are larger than pancreatin, they are not easily filtered, so they can be filtered by a filter. Divide into 10ml vials and keep at -20 degrees c.. (11) gelatin solution: Since gelatin is difficult to filter, preparation of the 0.1% gelatin solution must be made with sterile PBS. Therefore, the preparation process must pay attention to aseptic operation. The first problem is how to accurately weigh 0.1 grams (100ml solution) - that is, to solve the problem of aseptic dispensing. Second, it should be noted that even the solution of 0.1%, the gelatin is difficult to dissolve, so it should be fully shaken, overnight placed, and then divided into small bottles of 50ml, 4 degrees preservation. (12) preparation of other culture fluids: 20ug/ml endothelial growth factor Matters needing attention: 1, the preparation of the solution must use fresh distilled water. 2, the installation of CAI filters usually use pore size 0.45 microns and 0.22 microns filter, each place 0.45 placed in the 0.22 micron filter membrane above, and pay special attention to the filter face up. 3, preparation of RPMI1640 medium for add in calf serum, calf serum and slightly acidic medium, in order to ensure the final pH value is 7.2, in the preparation of Shidiao PH to 7.4.