Equipment and reagents
Dry powder medium, trypsin, penicillin, streptomycin. Purified water system, electronic balance, PH meter, magnetic stirrer, etc.
Specific steps
1. Water:
The cell culture water must be very pure and free of ions and other impurities. Fresh double distilled water, triple distilled water or purified water is required.
2. PBS (can also be used for the preparation of other BSS, such as: Hanks, D-Hanks solution):
Mainly used for rinsing tissues or cells during immunohistochemical staining
Dissolve to constant volume: Pour the medicine (NaCl 8.0g, KCl 0.2g, Na2HPO4% 26middot; H2O 1.56g, KH2PO4 0.2g) into a beaker with double distilled water, stir it with a glass rod to dissolve it fully, then pour the solution Place it in a volumetric flask and make sure to make up to 1000ml, shake it to make a freshly prepared PBS solution. Adjust the pH to 7.4 with HCl or NaOH.
Transfer into the solution bottle for sterilization: Pour PBS into the solution bottle (large needle bottle), cover with a rubber cap, and insert the needle into the 8-pound autoclave for sterilization for 20 minutes. Note that after autoclaving, use sterile distilled water to replenish the evaporated water.
3. Trypsin solution:
The role of trypsin is to hydrolyze the proteins between cells to separate the cells. Different tissues or cells respond differently to pancreatic enzymes. The activity of pancreatin dispersing cells is also related to its concentration, temperature and action time. At a pH of 8.0 and a temperature of 37 ° C, the action ability of the pancreatin solution is the strongest. When using pancreatin, the concentration, temperature and time should be grasped to avoid cell damage caused by excessive digestion. Since Ca2 +, Mg2 + and serum and protein can reduce the activity of pancreatin, BSS without Ca2 + and Mg2 + should be used when preparing pancreatin solution, such as D-Hanks solution. When the digestion is stopped, the effect of pancreatin on the cells can be terminated with serum culture medium or pancreatin inhibitor.
Weighing trypsin: According to the concentration of trypsin solution is 0.25%, use an electronic balance to accurately weigh the double-distilled water dissolved in a small beaker (if you use double-distilled water to adjust the pH to about 7.2) or PBS (D-hanks) In the liquid. Stir and mix and place at 4 ° C overnight.
Sterilization by suction filtration with an injection filter: the prepared pancreatin solution should be filtered and sterilized with an injection filter (0.22 micron microporous filter membrane) in the ultra-clean table. Then dispense into vials and store at -20 ° C for use.
4. 0.05% trypsin-0.02% EDTA solution
Weigh 0.05g of trypsin powder (1: 250), 0.02g of EDTA, add 100ml of PBSA, 0.22um microporous filter membrane to filter and sterilize, divide into pieces, and store at -20 ℃.
5. Green and streptomycin solution:
The pure water used (double distilled water) requires 15 pounds of high pressure for 20 minutes for sterilization. The specific operations are completed in the ultra-clean platform. Penicillin is 800,000 units / bottle, add 4ml of sterile double distilled water with a syringe. Streptomycin is 1 million units / bottle, plus 5ml of sterilized double distilled water, that is 200,000 units per ml. Store in aliquots at -20 ° C. When used, it is dissolved in the culture solution so that the concentration of penicillin is finally 100 units / ml.
6. RPMI1640:
Dissolution, pH adjustment, constant volume: First add the culture medium powder to the double distilled water of 2/3 of the volume of the culture liquid, and rinse the packaging bag with the double distilled water 2-3 times (the rinse liquid is added to the culture medium at the same time), and stir thoroughly After the powder is completely dissolved, add certain drugs according to the packaging instructions. Then add 0.5ml of the prepared penicillin solution to the culture medium with a syringe to make the concentration of penicillin 100 units / ml. Then adjust the pH to around 7.2 with carbon dioxide or sodium bicarbonate. Finally, make up to 1000ml and shake well.
Install the Cai filter: first install the bracket, install the filter membrane according to the regulations, and connect the stainless steel filter and the bracket with screws. Then remove the legs of the stand and wrap them with cloth to be sterilized.
Suction filtration: The prepared culture fluid is usually filtered and sterilized with a filter. Usually use a Tsai filter to filter in the ultra-clean workbench.
Dispensing: Dispense the filtered culture solution into a vial and put it in a 4 ℃ refrigerator for use.
Before use, add 1ml of glutamine solution to 100ml culture solution (effective at 4 ℃ for two weeks).
7. Inactivation of serum:
The commonly used cell culture is calf serum. The newly purchased serum must be inactivated in a 56 ° C water bath for 30 minutes, and then it can be added to the culture medium after suction filtration.
8. HEPES solution:
The chemical full name of HEPES is hydroxyethylpyrazine ethanesulfonic acid (N% 26rsquo; -a-hydroxythylpiperazine-N% 26rsquo; -ethanesulfanic acid). No toxic effects on cells. It is a hydrogen ion buffer that can control a constant pH range for a longer period of time. The final concentration is 10-50mmol / L, and the general culture medium contains 20mmol / L HEPES to achieve buffering capacity.
The preparation method of 1mol / L HEPE buffer is as follows:
Accurately weigh 238.3g of HEPTS, and add fresh three distilled water to bring the volume to 1L. 0.22um microporous filter membrane is sterilized by filtration, and stored at 4 ℃ after being packed.
Note: Because the commercially available HEPES is about 10g small vials, it can be flexibly formulated according to the actual situation, but it must be ensured that the final concentration of HEPES in the culture medium is still 20m mol / L. For example: Weigh 4.766 grams of HEPES dissolved in 20ml of three distilled water, after filtration and sterilization, it can be completely (20ml) added to 1L culture medium, or 2ml per 100ml culture medium.
9. Glutamine:
Synthetic medium contains a large amount of glutamine, and its role is very important. Cells need glutamine to synthesize nucleic acids and proteins. Lack of glutamine can cause poor cell growth or even death. A certain amount of glutamine should be added in the preparation of various culture solutions. Because glutamine is very unstable in the solution, it can be decomposed by 50% for one week at 4 ° C, so it should be prepared separately and stored in a -20 ° C refrigerator. When the culture solution added with glutamine is stored in a refrigerator at 4 ° C for more than 2 weeks, the original glutamine should be added again.
Generally, the content of glutamine in the culture medium is 1 to 4 mmol / L. Can prepare 200mmol / L glutamine liquid for storage, add culture medium when used. The preparation method is that 2.922g of glutamine is dissolved in triple distilled water and added to 100ml to prepare a solution of 200mmol / L. After fully stirred and dissolved, it is filtered and sterilized, divided into vials, stored at -20 ℃, and can be cultured in 100ml when used Add 1ml of glutamine solution to the solution.
10. Preparation of heparin solution:
The culture fluid containing heparin can improve the purity of endothelial cells. The final concentration of heparin added to the whole culture fluid is 50ug / ml. Because most of the heparin sodium is commercially available, the packaging is about 0.56 g / bottle. When preparing, it can be dissolved in 100ml of three distilled water, set the volume overnight, then filter and sterilize, divide the vial, and store the temperature at- -℃. When using, add 1ml (accurately 0.9ml) to 100ml culture solution.
11. Type â… collagenase:
The 0.1% type I collagenase solution is prepared and sterilized like trypsin. Note: Because the type â… collagenase molecular particles are larger than pancreatin, it is not easy to filter, so you can use a Cai filter to filter and sterilize. Divide into 10ml vials and store at -20 ℃.
12. Gelatin solution:
Because gelatin is difficult to filter, a 0.1% gelatin solution must be prepared with sterile PBS. Therefore, attention must be paid to aseptic operation during the preparation process. The first problem is how to accurately weigh 0.1 g (made into a 100ml solution)% 26mdash; that is, to solve the problem of aseptically dispensing medicines. Secondly, it should be noted that even the solution of 01., the gelatin is also difficult to dissolve, so it should be shaken well, placed overnight, and then aseptically divided into 50ml vials and stored at 4 ℃.
13. Hanks solution:
Weigh KH2PO4 0.06g, Nacl 8.0g, NaHCO3 0.35g, KCl 0.4g, glucose 1.0g, Na2HPO4.H2O 0.06g, add H2O to 1000ml
Note: Hanks solution can be autoclaved. Store in aliquots at 4 ° C.
14. D-hanks solution:
1 liquid: NaCL: 8.00g / L, KCL: 0.04g / L, Na2HPO4.2H2O: 0.06g / L, KH2PO4: 0.06g / L, with 500ml;
2 liquids: NaHCO3: 0.35g / L, with 100ml;
3 liquids: phenol red: 0.02g, dissolve phenol red with a few drops of NaHCO3
Transfer 2, 3 liquid into 1 liquid. The constant volume to 1000 ml PH value is about 7.4. Store in aliquots at 4 ° C.
15. Giemsa dye solution:
Weigh 0.5 g of Giemsa powder, grind with a few drops of glycerin, and then add glycerin (to make the total amount of added glycerin 33 ml). Incubate at 56 ° C for 90 to 120 minutes. Add 33ml of methanol and store in a brown bottle. This is Giemsa stock solution. Dilute with PBS as required. Generally diluted 10 times.
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