OptiPrep说明书（北京德博生物）

PRODUCT DESCRIPTION OptiPrep™ is a ready made, sterile and endotoxin tested solution of Iodixanol, 5,5’-[(2-hydroxy-1-3 propanediyl)-bis(acetylamino)] bis [N,N’- bis(2,3dihydroxypropyl-2,4,6-triiodo-1,3-benzenecarboxamide], desig- ned for the in vitro isolation of biological particles. Composition Iodixanol: 60% (w/v) in water Physical-chemical characteristics: Density: 1.320 ± 0.001 g/ml (20°C) Osmolality: 170 ± 15 mOsm OPTIPREP™ WORKING AND GRADIENT SOLUTIONS FOR CELLS An illustrative example of preparing working solutions for cells, based on a cell suspension medium (CSM) containing 0.85% (w/v) NaCl, 10 mM Tricine-NaOH, pH 7.4, is given in Table 1. Table 2 describes the density of gradient solutions prepared by dilution of the working solution with CSM. Hepes-buffered media provide solutions of identical densities and refractive indices. Low concentrations of additives (e.g. 1-5 mM MgCl2 or 1-2 mM EDTA) can be included without significant effect on the density or osmolality of the solutions. Alternatively, isoosmotic solutions can be prepared simply by diluting OptiPrep™ with a buffered 0.85 (w/v) NaCl or a routine culture medium such as DMEM or RPMI. All these media (in the absence of added serum) have densities of 1.006-1.007 g/ml (Table 3). A culture medium containing 10% serum has a slightly higher density at approx 1.009 g/ml. OPTIPREP™ WORKING AND GRADIENT SOLUTIONS FOR MAMMALIAN ORGANELLES Table 4 describes the preparation of a general purpose Working Solution for mammalian organelles and also modifications for mitochondria and nuclei. Other minor variations such as omission of the EDTA or inclusion of 0.1% (v/v) ethanol or 1-5 mM DTT, or use of alternative buffers will not affect the density of osmolality of the solutions. 621. Mar. 03. Div. 1114739 OptiPrep™ Table 1 OptiPrep™ Working Solutions for mammalian cells Cell suspension medium (CSM): 0.85% (w/v) NaCl, 10 mM Tricine-NaOH, pH 7.4 (ρ = 1.006 g/ml) To prepare a 40% (w/v) iodixanol Working Solution (ρ = 1.215 g/ml; η = 1.3976): Diluent required: 0.85% (w/v) NaCl, 30 mM Tricine-NaOH, pH 7.4. Mix 2 vol of OptiPrep™ with 1 vol of diluent. To prepare a 30% (w/v) iodixanol Working Solution (ρ = 1.163 g/ml; η = 1.3820): Diluent required: 0.85% (w/v) NaCl, 20 mM Tricine-NaOH, pH 7.4. Mix 1 vol of OptiPrep™ with 1 vol of diluent. Dilute Working Solution with CSM to make gradient solutions (Table 2) Table 2 Properties of iodixanol-NaCl/Tricine solutions1 % iodixanol (w/v) η ρ (g/ml) 40% iodixanol WS (ml) CSM (ml) 6.00 1.3444 1.037 0.6 3.4 8.00 1.3475 1.048 0.8 3.2 10.00 1.3507 1.058 1.0 3.0 12.00 1.3538 1.069 1.2 2.8 14.00 1.3569 1.079 1.4 2.6 16.00 1.3601 1.090 1.6 2.4 18.00 1.3632 1.100 1.8 2.2 20.00 1.3663 1.111 2.0 2.0 22.00 1.3694 1.121 2.2 1.8 24.00 1.3726 1.132 2.4 1.6 26.00 1.3757 1.142 2.6 1.4 28.00 1.3788 1.153 2.8 1.2 30.00 1.3820 1.163 3.0 1.0 32.00 1.3851 1.174 3.2 0.8 34.00 1.3882 1.184 3.4 0.6 36.00 1.3914 1.195 3.6 0.4 38.00 1.3945 1.205 3.8 0.2 40.00 1.3976 1.215 4.0 0 1 WS = working solution; CSM = cell suspension medium, η = refractive index; ρ = density The osmolality of the gradient solutions is in the range 285-300 mOsm. Table 3 Properties of iodixanol-DMEM solutions1 % iodixanol (w/v) η ρ (g/ml) 40% iodixanol WS (ml) DMEM(ml) 6.00 1.3448 1.038 0.6 3.4 8.00 1.3479 1.049 0.8 3.2 10.00 1.3510 1.059 1.0 3.0 12.00 1.3541 1.070 1.2 2.8 14.00 1.3572 1.080 1.4 2.6 16.00 1.3603 1.090 1.6 2.4 18.00 1.3633 1.101 1.8 2.2 20.00 1.3664 1.111 2.0 2.0 22.00 1.3695 1.122 2.2 1.8 24.00 1.3726 1.132 2.4 1.6 26.00 1.3757 1.143 2.6 1.4 28.00 1.3788 1.153 2.8 1.2 30.00 1.3819 1.163 3.0 1.0 32.00 1.3850 1.174 3.2 0.8 34.00 1.3881 1.184 3.4 0.6 36.00 1.3912 1.195 3.6 0.4 38.00 1.3943 1.205 3.8 0.2 40.00 1.3974 1.216 4.0 0 1 WS = working solution (4 vol of OptiPrep™ + 2 vol DMEM), η = refractive index; ρ = density The osmolality of the gradient solutions is in the range 290-305 mOsm. Table 4 OptiPrep™ Working Solutions for mammalian organelles Homogenization media (HM): General purpose: 0.25 M sucrose, 1 mM EDTA 10 mM Tris-HCl, pH 7.4 (ρ = 1.030 g/ml) Nuclei: 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.8 (ρ = 1.033 g/ml) Mitochondria: 4.4% (w/v) mannitol, 1 mM EDTA 10 mM Tris-HCl, pH 7.4 (ρ = 1.015 g/ml) To prepare a 50% (w/v) iodixanol Working Solution (WS1-3): Mix 5 vol of OptiPrep‘ with 1 vol of one of the following diluents: General purpose: 0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl, pH 7.4. (ρ = 1.032 g/ml) Nuclei: 150 mM KCl, 30 mM MgCl2, 120 mM Tris-HCl, pH 7.8 (ρ = 1.012 g/ml) Mitochondria: 4.4% (w/v) mannitol, 6 mM EDTA 60 mM Tris-HCl, pH 7.4 (ρ = 1.017 g/ml) WS1: ρ = 1.272 g/ml; η = 1.4147 WS2: ρ = 1.269 g/ml; η = 1.4148 WS3: ρ = 1.269 g/ml; η = 1.4139 Dilute WS with appropriate HM to make gradient solutions (Tables 5-7) Table 5 Properties of iodixanol-sucrose solutions1 % iodixanol (w/v) η ρ (g/ml) 50% iodixanol WS1 (ml) HM1(ml) 6.00 1.3534 1.059 1.2 8.8 8.00 1.3562 1.069 1.6 8.4 10.00 1.3589 1.079 2.0 8.0 12.00 1.3617 1.088 2.4 7.6 14.00 1.3645 1.098 2.8 7.2 16.00 1.3673 1.107 3.2 6.8 18.00 1.3701 1.117 3.6 6.4 20.00 1.3729 1.127 4.0 6.0 22.00 1.3757 1.136 4.4 5.6 24.00 1.3785 1.146 4.8 5.2 26.00 1.3813 1.156 5.2 4.8 28.00 1.3840 1.165 5.6 4.4 30.00 1.3868 1.175 6.0 4.0 32.00 1.3896 1.185 6.4 3.6 34.00 1.3924 1.194 6.8 3.2 36.00 1.3952 1.204 7.2 2.8 38.00 1.3980 1.214 7.6 2.4 40.00 1.4008 1.223 8.0 2.0 42.00 1.4036 1.233 8.4 1.6 44.00 1.4064 1.243 8.8 1.2 46.00 1.4091 1.252 9.2 0.8 48.00 1.4119 1.262 9.6 0.4 50.00 1.4147 1.272 1 WS = working solution, HM = homogenization medium; η = refractive index; ρ = density The osmolality of the gradient solutions is in the range 295-310 mOsm. OPTIPREP™ WORKING AND GRADIENT SOLUTIONS FOR NON-MAMMALIAN ORGANELLES Media containing 400-600 mM mannitol or sorbitol are common but for yeast organelles. The relationship between osmolality and concentration of sorbitol solutions in water is given in Table 8. The general strategy for production of gradient solutions of the correct osmolality is described in Tables 9-11. For more information see “Solutions for the formation of gradients” in the main Axis-Shield Catalogue “Applications and Products” STABILITY AND STORAGE OptiPrep™ is stable for 3 years provided the solution is kept sterile and protected from light. Prolonged exposure to direct sunlight leads to re- lease of iodine from the molecule. This effect is negligible when working with these solutions on a day to day basis. OptiPrep™ should be stored between 4°C - 24°C. Before use invert the bottles several times to mix the contents. ORDERING INFORMATION OptiPrep™ prod. no 1114542 1 x 250 ml Manufacturer: AXIS-SHIELD PoC AS P.O. Box 6863 Rodeløkka N-0504 Oslo, Norway Phone: +47 22 04 20 00 Fax: +47 22 04 20 01 www.axis-shield-poc.com ISO 9001 and ISO 13485 certified company Table 6 Properties of iodixanol-sucrose-KCl-MgCl2 solutions1 % iodixanol (w/v) η ρ (g/ml) 50% iodixanol WS2 (ml) HM2(ml) 10.0 1.3604 1.080 2.0 8.0 20.0 1.3740 1.127 4.0 6.0 30.0 1.3876 1.175 6.0 4.0 40.0 1.4012 1.222 8.0 2.0 50.0 1.4148 1.269 1 WS = working solution, HM = homogenization medium; η = refractive index; ρ = density The osmolality of the gradient solutions is in the range 320-360 mOsm. Table 7 Properties of iodixanol-mannitol solutions1 % iodixanol (w/v) η ρ (g/ml) 50% iodixanol WS3 (ml) HM3(ml) 10.0 1.3548 1.065 2.0 8.0 20.0 1.3796 1.116 4.0 6.0 30.0 1.3844 1.167 6.0 4.0 40.0 1.3991 1.218 8.0 2.0 50.0 1.4139 1.269 1 WS = working solution, HM = homogenization medium; η = refractive index; ρ = density The osmolality of the gradient solutions is in the range 292-310 mOsm. Table 8 Density (ρ), refractive index (η) and osmolality (σ) of sorbitol solutions in 10 mM Tris-HCl, pH 7.4 % sorbitol (w/v) η ρ (g/ml) σ (mOsm) 4.40 1.3390 1.015 265 8.75 1.3455 1.029 525 10.50 1.3480 1.035 657 12.25 1.3505 1.041 774 17.50 1.3580 1.059 1200 Table 9 OptiPrep™ Working Solutions (sorbitol) for non-mammalian organelles A 550 mOsm Working Solution Suspension medium (SMA): 8.75% (w/v) sorbitol, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4 To prepare a 40% (w/v) iodixanol Working Solution (WSA) Diluent required: 12.25% sorbitol, 3 mM EDTA, 30 mM Tris-HCl, pH 7.4. Mix 2 vol of OptiPrep™ with 1 vol of diluent Properties of 40% iodixanol WSA ρ = 1.225 g/ml; η = 1.4020; σ = 545 mOsm. Dilute WSA with SMA to make solutions of lower densities (Table 10) B 750 mOsm Working Solution Suspension medium (SMB): 12.25% (w/v) sorbitol, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4 To prepare a 40% (w/v) iodixanol Working Solution (WSB) Diluent required: 17.5% sorbitol, 3 mM EDTA, 30 mM Tris-HCl, pH 7.4. Mix 2 vol of OptiPrep™ with 1 vol of diluent Properties of 40% iodixanol WSB ρ = 1.231 g/ml; η = 1.4032; σ = 756 mOsm. Dilute WSB with SMB to make solutions of lower densities (Table 11) Table 10 Properties of iodixanol-sorbitol gradient solutions (approx 550 mOsm)1 % iodixanol (w/v) η ρ (g/ml) 40% iodixanol WSA (ml) SMA (ml) 6.00 1.3536 1.059 0.6 3.4 8.00 1.3565 1.068 0.8 3.2 10.00 1.3596 1.078 1.0 3.0 12.00 1.3622 1.088 1.2 2.8 14.00 1.3650 1.098 1.4 2.6 16.00 1.3679 1.108 1.6 2.4 18.00 1.3707 1.117 1.8 2.2 20.00 1.3736 1.127 2.0 2.0 22.00 1.3764 1.137 2.2 1.8 24.00 1.3792 1.147 2.4 1.6 26.00 1.3821 1.157 2.6 1.4 28.00 1.3849 1.166 2.8 1.2 30.00 1.3878 1.176 3.0 1.0 32.00 1.3906 1.186 3.2 0.8 34.00 1.3935 1.196 3.4 0.6 36.00 1.3963 1.205 3.6 0.4 38.00 1.3992 1.215 3.8 0.2 40.00 1.4020 1.225 4.0 0 1 WS = working solution, SM = suspension medium; η = refractive index; ρ = density The osmolality of the gradient solutions is in the range 545-560 mOsm. Table 11 Properties of iodixanol-sorbitol gradient solutions (approx 750 mOsm)1 % iodixanol (w/v) η ρ (g/ml) 40% iodixanol WSB (ml) SMB (ml) 6.00 1.3584 1.070 0.6 3.4 8.00 1.3610 1.079 0.8 3.2 10.00 1.3637 1.089 1.0 3.0 12.00 1.3663 1.098 1.2 2.8 14.00 1.3689 1.108 1.4 2.6 16.00 1.3716 1.117 1.6 2.4 18.00 1.3742 1.127 1.8 2.2 20.00 1.3769 1.136 2.0 2.0 22.00 1.3795 1.146 2.2 1.8 24.00 1.3821 1.155 2.4 1.6 26.00 1.3848 1.165 2.6 1.4 28.00 1.3874 1.174 2.8 1.2 30.00 1.3900 1.184 3.0 1.0 32.00 1.3927 1.193 3.2 0.8 34.00 1.3953 1.203 3.4 0.6 36.00 1.3979 1.212 3.6 0.4 38.00 1.4006 1.222 3.8 0.2 40.00 1.4032 1.231 4.0 0 1 WS = working solution, SM = suspension medium; η = refractive index; ρ = density The osmolality of the gradient solutions is in the range 755-850 mOsm.