Instructions for dissolving AppliChem powder media to prepare sterile liquid cell culture media

Transcription

1 Instructions for dissolving AppliChem powder media to prepare sterile liquid cell culture media General Information Powdered media and salts are very hygroscopic and must be stored under dry conditions. After opening the package the whole contents must be dissolved at once. Reconstitute the powdered form of media to produce 1X liquid medium, as the different amino acids may precipitate because of their low solubility. They potentially can form salts which are of low solubility in concentrated solutions. If supplements are needed, they can be added before filtration (unsterile) or after filtration (sterile). Use bidistilled or deionized, pyrogen-free water to reconstitute powder media. Procedure 1: Preparing sterile filtered liquid media completely dissolved. The temperature of the water should be between C 2.) When the powder is completely dissolved, add sodium hydrogen carbonate and dissolve completely also. The required quantities are listed in the table below. 3.) Adjust to the desired ph value (physiological optimum is ph ) with 1 N HCl or 1 N NaOH while stirring. Note: The ph should be approx. 0.2 units lower than the target ph, because the ph will rise slightly during filtration, when CO 2 leaks out. 4.) After adjusting the ph, add water to the appropriate final volume and mix well. Filter immediately under sterile conditions. 5.) Store the medium at 2-8 C protected from light. Procedure 2: Preparing liquid media from autoclavable powder media completely dissolved. The temperature of the water should be between C 2.) Check the ph value of the solution. The ph value should be ca If necessary adjust with 1 N HCl solution. 3.) Prepare aliquots of 500 ml max. The lids should only be opened a little to avoid contamination during autoclaving. Autoclave 15 min at 121 C. 4.) After autoclaving let the medium cool down to room temperature. 5.) Under sterile conditions add a sterile-filtered solution of 7.5 % sodium hydrogen carbonate. Add the volume according to the table below. Note: Add sterile glutamine, serum and other heat sensitive additives (e.g. antibiotics or growth factors) after autoclaving. 6.) Check the ph value of the solution. The physiological optimum for mammalian cells is ph If necessary adjust the ph value with sterile solutions of 1 N HCl or 1 N NaOH. 7.) After adjusting the ph add sterile water to the final volume and mix well. 8.) Store the medium at 2-8 C protected from light. 1

2 Procedure 3: Preparing liquid insect cell culture media completely dissolved. The temperature of the water should be between C. 2.) When the powder is completely dissolved, add sodium hydrogen carbonate and also allow to dissolve completely. The required quantities are listed in the table below. 3.) Adjust to ph 6.3* with 1 N HCl or 1 N NaOH while stirring. Note: The ph should be approx. 6,1, i.e. 0.2 units lower than the target ph, because the ph will slightly rise during filtration when the CO 2 leaks out. *Comment: The physiological ph optimum of insect cell culture media is more acidic compared to the ph of mammalian cell culture media. At ph values above approx. 6.5, an undesired precipitation will occur with insect cell culture media which contain a high concentration of calcium chloride. 4.) After adjusting the ph, add water to the final volume. Mix well and filter immediately. 5.) Store the medium at 2-8 C protected from light. 2

3 Table for the addition of sodium hydrogen carbonate BME, Basal medium with Earle's salts A BME, Basal medium with Earle's salts A X BME, Basal medium with Earle's salts A / L 1X 10X BME, Basal medium with Earle's salts A / L 1X BME, Basal medium with Earle's salts, with HEPES A BME, Basal medium with Earle's salts, with HEPES A BME, Basal medium with Earle's (diploide) A BME, Basal medium with Earle's (diploide) A BME, Basal medium with Hanks' salts A BME, Basal medium with Hanks' salts A X BME, Basal medium with Hanks' salts A / L 1X 10X BME, Basal medium with Hanks' salts A / L 1X BME, Basal medium with Hanks' salts; autoclavable A ml 7.5 % - Solution / L after autoclaving BME, Basal medium with Hanks' salts; autoclavable A ml 7.5 % - Solution / L after autoclaving BME, Basal medium with Hanks' salts, with HEPES A BME, Basal medium with Hanks' salts, with HEPES A DMEM - Medium A DMEM - Medium A DMEM - Medium with HEPES A DMEM - Medium with HEPES A DMEM - Medium A DMEM - Medium A DMEM - Medium A DMEM - Medium A DMEM - Medium with HEPES A DMEM - Medium with HEPES A DMEM - Medium with HEPES A DMEM - Medium with HEPES A DMEM - Medium A DMEM - Medium A DMEM - Medium A DMEM - Medium A DMEM/F12 (1:1) - Medium A DMEM/F12 (1:1) - Medium A DMEM/F12 (1:1) - Medium with 15 mm HEPES A DMEM/F12 (1:1) - Medium with 15 mm HEPES A DMEM/F12 (1:1) - Medium with 25 mm HEPES A DMEM/F12 (1:1) - Medium with 25 mm HEPES A Earle's balanced salts A Earle's balanced salts A Earle's balanced salts A Earle's balanced salts A Gamborg's B5 - Plant salts A1911 none Glasgow - MEM (BHK 21) A Glasgow - MEM (BHK 21) A Glasgow - MEM (BHK 21) A Glasgow - MEM (BHK 21) A Grace's - Insect medium A Grace's - Insect medium A Ham's F10 - Medium A Ham's F10 - Medium A Ham's F10 - Medium with HEPES A Ham's F10 - Medium with HEPES A Ham's F10 - Medium with HEPES A Ham's F10 - Medium with HEPES A Ham's F12K - Medium A Ham's F12K - Medium A Ham's F12 - Medium A Ham's F12 - Medium A Ham's F12 - Medium with HEPES A Ham's F12 - Medium with HEPES A Ham's F12K - Medium A Ham's F12K - Medium A

4 Table for the addition of sodium hydrogen carbonate (continued) Hanks' balanced salts A Hanks' balanced salts A Hanks' balanced salts A Hanks' balanced salts A HEPES balanced salts A0629 none Iscove's mod. Dulbecco's - Medium (IMDM) A Iscove's mod. Dulbecco's - Medium (IMDM) A Iscove's mod. Dulbecco's - Medium (IMDM) A Iscove's mod. Dulbecco's - Medium (IMDM) A IPL 41 - Medium A IPL 41 - Medium A Joklik - MEM, modified for spinner culture A Joklik - MEM, modified for spinner culture A Leibovitz's L-15 - Medium A1967 none Leibovitz's L-15 - Medium A1323 none Leibovitz's L-15 - Medium with HEPES A1968 none Leibovitz's L-15 - Medium with HEPES A1954 none Linsmaier, Bednar & Skoog Plant salts A0591 none McCoy's 5A - Medium (modified) A McCoy's 5A - Medium (modified) A McCoy's 5A - Medium (modified) A McCoy's 5A - Medium (modified) A Medium 199 with Earle's salts A Medium 199 with Earle's salts A Medium 199 with Earle's salts, with HEPES A Medium 199 with Earle's salts, with HEPES A Medium 199 with Hanks' salts A Medium 199 with Hanks' salts A Medium 199 with Hanks' salts, with HEPES A Medium 199 with Hanks' salts, with HEPES A a - MEM A a - MEM A a - MEM A a - MEM A a - MEM with HEPES A a - MEM with HEPES A a - MEM with HEPES A a - MEM with HEPES A MEM with Earle's salts A MEM with Earle's salts A MEM with Earle's salts A MEM with Earle's salts A MEM with Earle's salts, with HEPES A MEM with Earle's salts, with HEPES A MEM with Earle's salts, with HEPES A MEM with Earle's salts, with HEPES A MEM with Earle's salts; autoclavable A ml 7.5 % - Solution / L after autoclaving MEM with Hanks' salts A MEM with Hanks' salts A MEM with Hanks' salts A MEM with Hanks' salts A MEM with Hanks' salts, with HEPES A MEM with Hanks' salts, with HEPES A MEM with Hanks' salts, with HEPES A MEM with Hanks' salts, with HEPES A S-MEM, modified for spinner culture A S-MEM, modified for spinner culture A S-MEM, modified for spinner culture; autoclavable A ml 7.5 % - Solution / L after autoclaving Murashige & Skoog - Plant salts A1534 none Murashige & Skoog - Plant salts A1535 none 4

5 Table for the addition of sodium hydrogen carbonate (continued) NCTC Medium A NCTC Medium A PBS buffer (1X, Dulbecco) - Powder A0964 none PBS buffer (10X, Dulbecco) - Powder A0965 none PBS buffer (1X, Dulbecco) - Powder A1908 None RPMI 1640 A RPMI 1640 A RPMI 1640 with HEPES A RPMI 1640 with HEPES A RPMI 1640 A RPMI 1640 A Click-RPMI A Click-RPMI A Schneider's Drosophila - Insect medium A Schneider's Drosophila - Insect medium A TC Medium A TC Medium A TNM-FH - Insect medium A TNM-FH - Insect medium A Tris - Citrate balanced salts A0692 none Waymouth's MB 752/1 - Medium A Waymouth's MB 752/1 - Medium A Waymouth's MB 752/1 - Medium with 20 mm HEPES A Waymouth's MB 752/1 - Medium with 20 mm HEPES A William's Medium E A William's Medium E A William's Medium E with HEPES A William's Medium E with HEPES A William's Medium E A William's Medium E A version:mm