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 15-30 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 6.8-7.2) 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 15-30 C 2.) Check the ph value of the solution. The ph value should be ca. 4.1. 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 6.8-7.2. 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
Procedure 3: Preparing liquid insect cell culture media completely dissolved. The temperature of the water should be between 15-30 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
Table for the addition of sodium hydrogen carbonate BME, Basal medium with Earle's salts A1310 2.200 BME, Basal medium with Earle's salts A1311 2.200 10X BME, Basal medium with Earle's salts A1977 2.200 / L 1X 10X BME, Basal medium with Earle's salts A1983 2.200 / L 1X BME, Basal medium with Earle's salts, with HEPES A1915 2.200 BME, Basal medium with Earle's salts, with HEPES A1916 2.200 BME, Basal medium with Earle's (diploide) A1984 2.200 BME, Basal medium with Earle's (diploide) A1312 2.200 BME, Basal medium with Hanks' salts A1917 0.350 BME, Basal medium with Hanks' salts A1313 0.350 10X BME, Basal medium with Hanks' salts A1990 0.350 / L 1X 10X BME, Basal medium with Hanks' salts A1988 0.350 / L 1X BME, Basal medium with Hanks' salts; autoclavable A1976 30 ml 7.5 % - Solution / L after autoclaving BME, Basal medium with Hanks' salts; autoclavable A1978 5 ml 7.5 % - Solution / L after autoclaving BME, Basal medium with Hanks' salts, with HEPES A1918 0.350 BME, Basal medium with Hanks' salts, with HEPES A1919 0.350 DMEM - Medium A1924 3.700 DMEM - Medium A1314 3.700 DMEM - Medium with HEPES A1925 0.850 DMEM - Medium with HEPES A1315 0.850 DMEM - Medium A1926 3.700 DMEM - Medium A1316 3.700 DMEM - Medium A1985 3.700 DMEM - Medium A1986 3.700 DMEM - Medium with HEPES A1950 0.850 DMEM - Medium with HEPES A1965 0.850 DMEM - Medium with HEPES A1979 0.850 DMEM - Medium with HEPES A1951 0.850 DMEM - Medium A0826 3.700 DMEM - Medium A3134 3.700 DMEM - Medium A3137 3.700 DMEM - Medium A3139 3.700 DMEM/F12 (1:1) - Medium A1920 1.200 DMEM/F12 (1:1) - Medium A1318 1.200 DMEM/F12 (1:1) - Medium with 15 mm HEPES A1922 1.200 DMEM/F12 (1:1) - Medium with 15 mm HEPES A1317 1.200 DMEM/F12 (1:1) - Medium with 25 mm HEPES A1923 1.200 DMEM/F12 (1:1) - Medium with 25 mm HEPES A1921 1.200 Earle's balanced salts A1712 2.200 Earle's balanced salts A2039 2.200 Earle's balanced salts A3026 2.200 Earle's balanced salts A3127 2.200 Gamborg's B5 - Plant salts A1911 none Glasgow - MEM (BHK 21) A2024 2.750 Glasgow - MEM (BHK 21) A1320 2.750 Glasgow - MEM (BHK 21) A2025 2.750 Glasgow - MEM (BHK 21) A1321 2.750 Grace's - Insect medium A2023 0.350 Grace's - Insect medium A1529 0.350 Ham's F10 - Medium A1970 1.200 Ham's F10 - Medium A1525 1.200 Ham's F10 - Medium with HEPES A1989 1.200 Ham's F10 - Medium with HEPES A1982 1.200 Ham's F10 - Medium with HEPES A1952 1.200 Ham's F10 - Medium with HEPES A2026 1.200 Ham's F12K - Medium A1963 2.500 Ham's F12K - Medium A1927 2.500 Ham's F12 - Medium A1948 1.176 Ham's F12 - Medium A1526 1.176 Ham's F12 - Medium with HEPES A1969 1.176 Ham's F12 - Medium with HEPES A1953 1.176 Ham's F12K - Medium A1963 2.500 Ham's F12K - Medium A1927 2.500 3
Table for the addition of sodium hydrogen carbonate (continued) Hanks' balanced salts A1711 0.350 Hanks' balanced salts A3140 0.350 Hanks' balanced salts A2729 0.350 Hanks' balanced salts A2512 0.350 HEPES balanced salts A0629 none Iscove's mod. Dulbecco's - Medium (IMDM) A2028 3.700 Iscove's mod. Dulbecco's - Medium (IMDM) A1909 3.700 Iscove's mod. Dulbecco's - Medium (IMDM) A2029 3.024 Iscove's mod. Dulbecco's - Medium (IMDM) A1322 3.024 IPL 41 - Medium A2027 0.350 IPL 41 - Medium A1964 0.350 Joklik - MEM, modified for spinner culture A1974 2.000 Joklik - MEM, modified for spinner culture A1539 2.000 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) A2034 2.200 McCoy's 5A - Medium (modified) A1324 2.200 McCoy's 5A - Medium (modified) A2035 2.200 McCoy's 5A - Medium (modified) A1955 2.200 Medium 199 with Earle's salts A2022 2.200 Medium 199 with Earle's salts A1325 2.200 Medium 199 with Earle's salts, with HEPES A2030 0.850 Medium 199 with Earle's salts, with HEPES A1326 0.850 Medium 199 with Hanks' salts A2031 0.350 Medium 199 with Hanks' salts A1327 0.350 Medium 199 with Hanks' salts, with HEPES A2021 0.350 Medium 199 with Hanks' salts, with HEPES A1328 0.350 a - MEM A1971 2.200 a - MEM A1330 2.200 a - MEM A1913 2.200 a - MEM A1329 2.200 a - MEM with HEPES A1980 0.850 a - MEM with HEPES A1972 0.850 a - MEM with HEPES A1949 0.850 a - MEM with HEPES A1914 0.850 MEM with Earle's salts A1331 2.200 MEM with Earle's salts A1332 2.200 MEM with Earle's salts A2018 2.200 MEM with Earle's salts A1334 2.200 MEM with Earle's salts, with HEPES A2019 0.850 MEM with Earle's salts, with HEPES A1333 0.850 MEM with Earle's salts, with HEPES A2032 0.850 MEM with Earle's salts, with HEPES A1956 0.850 MEM with Earle's salts; autoclavable A2597 30 ml 7.5 % - Solution / L after autoclaving MEM with Hanks' salts A1957 0.350 MEM with Hanks' salts A1335 0.350 MEM with Hanks' salts A2041 0.350 MEM with Hanks' salts A1337 0.350 MEM with Hanks' salts, with HEPES A2042 0.350 MEM with Hanks' salts, with HEPES A1336 0.350 MEM with Hanks' salts, with HEPES A2043 0.350 MEM with Hanks' salts, with HEPES A1958 0.350 S-MEM, modified for spinner culture A1338 2.200 S-MEM, modified for spinner culture A1308 2.200 S-MEM, modified for spinner culture; autoclavable A1975 30 ml 7.5 % - Solution / L after autoclaving Murashige & Skoog - Plant salts A1534 none Murashige & Skoog - Plant salts A1535 none 4
Table for the addition of sodium hydrogen carbonate (continued) NCTC 135 - Medium A2020 2.200 NCTC 135 - Medium A1528 2.200 PBS buffer (1X, Dulbecco) - Powder A0964 none PBS buffer (10X, Dulbecco) - Powder A0965 none PBS buffer (1X, Dulbecco) - Powder A1908 None RPMI 1640 A1959 2.200 RPMI 1640 A1339 2.200 RPMI 1640 with HEPES A1960 0.850 RPMI 1640 with HEPES A1538 0.850 RPMI 1640 A2040 2.000 RPMI 1640 A3135 2.000 Click-RPMI A2044 1.175 Click-RPMI A2504 1.175 Schneider's Drosophila - Insect medium A2036 0.400 Schneider's Drosophila - Insect medium A1533 0.400 TC-100 - Medium A2033 0.350 TC-100 - Medium A2017 0.350 TNM-FH - Insect medium A2037 0.350 TNM-FH - Insect medium A1570 0.350 Tris - Citrate balanced salts A0692 none Waymouth's MB 752/1 - Medium A1973 2.240 Waymouth's MB 752/1 - Medium A1527 2.240 Waymouth's MB 752/1 - Medium with 20 mm HEPES A1981 0.850 Waymouth's MB 752/1 - Medium with 20 mm HEPES A1961 0.850 William's Medium E A1309 2.200 William's Medium E A1910 2.200 William's Medium E with HEPES A1966 0.850 William's Medium E with HEPES A1962 0.850 William's Medium E A3029 2.200 William's Medium E A3136 2.200 version:mm20101203 5