Modification:Article Title: Preparation of vitrified mammalian cells for in situ cryo-electron tomography
Article Snippet: .. INS-1E cells (gift of P. Maechler, Université de Genève) ( Merglen et al., 2004 ) Dulbecco's Modified Eagle Medium (DMEM) (Corning, #MT10013CV) Gibco RPMI 1640 Medium (Fisher Scientific, #1875119) Fetal bovine serum (FBS) (Corning, #35-010-CV) HEPES (1M) (Fisher Scientific Company, #15-630-080) Penicillin-streptomycin (10,000 U/mL) (Fisher Scientific Company, #15140122) Sodium pyruvate (100 mM) (Fisher Scientific, #11360070) L-Glutamine (200 mM) (Fisher Scientific, #25030081) 2-mercaptoethanol (Fisher Scientific, #21985023) Dulbecco’s Phosphate-Buffered Saline, 1X without calcium and magnesium (DPBS) 1x (Corning, # 21-031-CM) Trypsin-EDTA 1x (Corning, # 25-052-CI) Human fibronectin (Sigma, #F1056) Trypan Blue Solution, 0.4% (Fisher Scientific, #15250061) 70% Ethanol Mili-Q ddH20 CO 2 incubator Class II biosafety cabinet/Tissue culture (TC) laminar flow hood Chemical hood (for ethane and propane) Low-speed centrifuge (Eppendorf 5702 or equivalent) Hemocytometer or equivalent cell counter Inverted microscope for cell culture (Olympus or equivalent) Cell culture dishes 10cm, tissue culture treated (Corning, #CLS430599) 35 mm MatTek culture dishes, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated (MatTek, #P35G-1.5-14) Pipettes and pipette tips Parafilm (VWR, #3-374-5) Vitrobot Mark IV System (FEI/Thermo Fisher Scientific) and Vitrobot accessories ( i.e ., ethane tray, anticontamination ring, Vitrobot tweezers, holder for grid boxes, cooling spider, brass ethane cup) Whatman filter paper (GE Healthcare Life Sciences, #1001-055 and #1440-150) 60 mL syringe (Cole-Parmer, #0794542) Tweezers for EM grid handling (Dumont, #0203-L5-P0) Large tweezers for handling EM grid boxes (VWR, #82027-432) UltrAuFoil ® Gold R 2/2 on Gold 200 EM grids (Quantifoil/SPT Labtech, # N1-A16nAu20-01; alternative option: Electron Microscopy Sciences, #LFH2100AR2I) or alternatively QUANTIFOIL ® Carbon R 2/2 on Gold 200 (Quantifoil/SPT Labtech, #N1-C16nAu20-01; alternative option: Electron Microscopy Sciences, #Q2100AR2) Cryo grid box, round, with lid (Electron Microscopy Sciences, #71166-10-W) Screwdriver kit (Ted Pella, #53719) 350 mL Dewar shielded vacuum flask (Pope Scientific, #8600/0099) Liquid nitrogen (LN 2 ) storage unit for storing frozen samples ( e.g ., Thermo Fisher Scientific Locator cryogenic storage system) 15 mL and 50 mL conical tubes (Fisher Scientific, #14955237 and #1495949A) Kimwipes String, yard (to pull the samples out the liquid nitrogen) Liquid nitrogen (Matheson, #NIL160-22) Ethane (Matheson, #UN1035) Propane (Matheson, #UN1978) ..
Saline:Article Title: Preparation of vitrified mammalian cells for in situ cryo-electron tomography
Article Snippet: .. INS-1E cells (gift of P. Maechler, Université de Genève) ( Merglen et al., 2004 ) Dulbecco's Modified Eagle Medium (DMEM) (Corning, #MT10013CV) Gibco RPMI 1640 Medium (Fisher Scientific, #1875119) Fetal bovine serum (FBS) (Corning, #35-010-CV) HEPES (1M) (Fisher Scientific Company, #15-630-080) Penicillin-streptomycin (10,000 U/mL) (Fisher Scientific Company, #15140122) Sodium pyruvate (100 mM) (Fisher Scientific, #11360070) L-Glutamine (200 mM) (Fisher Scientific, #25030081) 2-mercaptoethanol (Fisher Scientific, #21985023) Dulbecco’s Phosphate-Buffered Saline, 1X without calcium and magnesium (DPBS) 1x (Corning, # 21-031-CM) Trypsin-EDTA 1x (Corning, # 25-052-CI) Human fibronectin (Sigma, #F1056) Trypan Blue Solution, 0.4% (Fisher Scientific, #15250061) 70% Ethanol Mili-Q ddH20 CO 2 incubator Class II biosafety cabinet/Tissue culture (TC) laminar flow hood Chemical hood (for ethane and propane) Low-speed centrifuge (Eppendorf 5702 or equivalent) Hemocytometer or equivalent cell counter Inverted microscope for cell culture (Olympus or equivalent) Cell culture dishes 10cm, tissue culture treated (Corning, #CLS430599) 35 mm MatTek culture dishes, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated (MatTek, #P35G-1.5-14) Pipettes and pipette tips Parafilm (VWR, #3-374-5) Vitrobot Mark IV System (FEI/Thermo Fisher Scientific) and Vitrobot accessories ( i.e ., ethane tray, anticontamination ring, Vitrobot tweezers, holder for grid boxes, cooling spider, brass ethane cup) Whatman filter paper (GE Healthcare Life Sciences, #1001-055 and #1440-150) 60 mL syringe (Cole-Parmer, #0794542) Tweezers for EM grid handling (Dumont, #0203-L5-P0) Large tweezers for handling EM grid boxes (VWR, #82027-432) UltrAuFoil ® Gold R 2/2 on Gold 200 EM grids (Quantifoil/SPT Labtech, # N1-A16nAu20-01; alternative option: Electron Microscopy Sciences, #LFH2100AR2I) or alternatively QUANTIFOIL ® Carbon R 2/2 on Gold 200 (Quantifoil/SPT Labtech, #N1-C16nAu20-01; alternative option: Electron Microscopy Sciences, #Q2100AR2) Cryo grid box, round, with lid (Electron Microscopy Sciences, #71166-10-W) Screwdriver kit (Ted Pella, #53719) 350 mL Dewar shielded vacuum flask (Pope Scientific, #8600/0099) Liquid nitrogen (LN 2 ) storage unit for storing frozen samples ( e.g ., Thermo Fisher Scientific Locator cryogenic storage system) 15 mL and 50 mL conical tubes (Fisher Scientific, #14955237 and #1495949A) Kimwipes String, yard (to pull the samples out the liquid nitrogen) Liquid nitrogen (Matheson, #NIL160-22) Ethane (Matheson, #UN1035) Propane (Matheson, #UN1978) ..
Inverted Microscopy:Article Title: Preparation of vitrified mammalian cells for in situ cryo-electron tomography
Article Snippet: .. INS-1E cells (gift of P. Maechler, Université de Genève) ( Merglen et al., 2004 ) Dulbecco's Modified Eagle Medium (DMEM) (Corning, #MT10013CV) Gibco RPMI 1640 Medium (Fisher Scientific, #1875119) Fetal bovine serum (FBS) (Corning, #35-010-CV) HEPES (1M) (Fisher Scientific Company, #15-630-080) Penicillin-streptomycin (10,000 U/mL) (Fisher Scientific Company, #15140122) Sodium pyruvate (100 mM) (Fisher Scientific, #11360070) L-Glutamine (200 mM) (Fisher Scientific, #25030081) 2-mercaptoethanol (Fisher Scientific, #21985023) Dulbecco’s Phosphate-Buffered Saline, 1X without calcium and magnesium (DPBS) 1x (Corning, # 21-031-CM) Trypsin-EDTA 1x (Corning, # 25-052-CI) Human fibronectin (Sigma, #F1056) Trypan Blue Solution, 0.4% (Fisher Scientific, #15250061) 70% Ethanol Mili-Q ddH20 CO 2 incubator Class II biosafety cabinet/Tissue culture (TC) laminar flow hood Chemical hood (for ethane and propane) Low-speed centrifuge (Eppendorf 5702 or equivalent) Hemocytometer or equivalent cell counter Inverted microscope for cell culture (Olympus or equivalent) Cell culture dishes 10cm, tissue culture treated (Corning, #CLS430599) 35 mm MatTek culture dishes, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated (MatTek, #P35G-1.5-14) Pipettes and pipette tips Parafilm (VWR, #3-374-5) Vitrobot Mark IV System (FEI/Thermo Fisher Scientific) and Vitrobot accessories ( i.e ., ethane tray, anticontamination ring, Vitrobot tweezers, holder for grid boxes, cooling spider, brass ethane cup) Whatman filter paper (GE Healthcare Life Sciences, #1001-055 and #1440-150) 60 mL syringe (Cole-Parmer, #0794542) Tweezers for EM grid handling (Dumont, #0203-L5-P0) Large tweezers for handling EM grid boxes (VWR, #82027-432) UltrAuFoil ® Gold R 2/2 on Gold 200 EM grids (Quantifoil/SPT Labtech, # N1-A16nAu20-01; alternative option: Electron Microscopy Sciences, #LFH2100AR2I) or alternatively QUANTIFOIL ® Carbon R 2/2 on Gold 200 (Quantifoil/SPT Labtech, #N1-C16nAu20-01; alternative option: Electron Microscopy Sciences, #Q2100AR2) Cryo grid box, round, with lid (Electron Microscopy Sciences, #71166-10-W) Screwdriver kit (Ted Pella, #53719) 350 mL Dewar shielded vacuum flask (Pope Scientific, #8600/0099) Liquid nitrogen (LN 2 ) storage unit for storing frozen samples ( e.g ., Thermo Fisher Scientific Locator cryogenic storage system) 15 mL and 50 mL conical tubes (Fisher Scientific, #14955237 and #1495949A) Kimwipes String, yard (to pull the samples out the liquid nitrogen) Liquid nitrogen (Matheson, #NIL160-22) Ethane (Matheson, #UN1035) Propane (Matheson, #UN1978) ..
Cell Culture:Article Title: Preparation of vitrified mammalian cells for in situ cryo-electron tomography
Article Snippet: .. INS-1E cells (gift of P. Maechler, Université de Genève) ( Merglen et al., 2004 ) Dulbecco's Modified Eagle Medium (DMEM) (Corning, #MT10013CV) Gibco RPMI 1640 Medium (Fisher Scientific, #1875119) Fetal bovine serum (FBS) (Corning, #35-010-CV) HEPES (1M) (Fisher Scientific Company, #15-630-080) Penicillin-streptomycin (10,000 U/mL) (Fisher Scientific Company, #15140122) Sodium pyruvate (100 mM) (Fisher Scientific, #11360070) L-Glutamine (200 mM) (Fisher Scientific, #25030081) 2-mercaptoethanol (Fisher Scientific, #21985023) Dulbecco’s Phosphate-Buffered Saline, 1X without calcium and magnesium (DPBS) 1x (Corning, # 21-031-CM) Trypsin-EDTA 1x (Corning, # 25-052-CI) Human fibronectin (Sigma, #F1056) Trypan Blue Solution, 0.4% (Fisher Scientific, #15250061) 70% Ethanol Mili-Q ddH20 CO 2 incubator Class II biosafety cabinet/Tissue culture (TC) laminar flow hood Chemical hood (for ethane and propane) Low-speed centrifuge (Eppendorf 5702 or equivalent) Hemocytometer or equivalent cell counter Inverted microscope for cell culture (Olympus or equivalent) Cell culture dishes 10cm, tissue culture treated (Corning, #CLS430599) 35 mm MatTek culture dishes, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated (MatTek, #P35G-1.5-14) Pipettes and pipette tips Parafilm (VWR, #3-374-5) Vitrobot Mark IV System (FEI/Thermo Fisher Scientific) and Vitrobot accessories ( i.e ., ethane tray, anticontamination ring, Vitrobot tweezers, holder for grid boxes, cooling spider, brass ethane cup) Whatman filter paper (GE Healthcare Life Sciences, #1001-055 and #1440-150) 60 mL syringe (Cole-Parmer, #0794542) Tweezers for EM grid handling (Dumont, #0203-L5-P0) Large tweezers for handling EM grid boxes (VWR, #82027-432) UltrAuFoil ® Gold R 2/2 on Gold 200 EM grids (Quantifoil/SPT Labtech, # N1-A16nAu20-01; alternative option: Electron Microscopy Sciences, #LFH2100AR2I) or alternatively QUANTIFOIL ® Carbon R 2/2 on Gold 200 (Quantifoil/SPT Labtech, #N1-C16nAu20-01; alternative option: Electron Microscopy Sciences, #Q2100AR2) Cryo grid box, round, with lid (Electron Microscopy Sciences, #71166-10-W) Screwdriver kit (Ted Pella, #53719) 350 mL Dewar shielded vacuum flask (Pope Scientific, #8600/0099) Liquid nitrogen (LN 2 ) storage unit for storing frozen samples ( e.g ., Thermo Fisher Scientific Locator cryogenic storage system) 15 mL and 50 mL conical tubes (Fisher Scientific, #14955237 and #1495949A) Kimwipes String, yard (to pull the samples out the liquid nitrogen) Liquid nitrogen (Matheson, #NIL160-22) Ethane (Matheson, #UN1035) Propane (Matheson, #UN1978) ..
Transferring:Article Title: Preparation of vitrified mammalian cells for in situ cryo-electron tomography
Article Snippet: .. INS-1E cells (gift of P. Maechler, Université de Genève) ( Merglen et al., 2004 ) Dulbecco's Modified Eagle Medium (DMEM) (Corning, #MT10013CV) Gibco RPMI 1640 Medium (Fisher Scientific, #1875119) Fetal bovine serum (FBS) (Corning, #35-010-CV) HEPES (1M) (Fisher Scientific Company, #15-630-080) Penicillin-streptomycin (10,000 U/mL) (Fisher Scientific Company, #15140122) Sodium pyruvate (100 mM) (Fisher Scientific, #11360070) L-Glutamine (200 mM) (Fisher Scientific, #25030081) 2-mercaptoethanol (Fisher Scientific, #21985023) Dulbecco’s Phosphate-Buffered Saline, 1X without calcium and magnesium (DPBS) 1x (Corning, # 21-031-CM) Trypsin-EDTA 1x (Corning, # 25-052-CI) Human fibronectin (Sigma, #F1056) Trypan Blue Solution, 0.4% (Fisher Scientific, #15250061) 70% Ethanol Mili-Q ddH20 CO 2 incubator Class II biosafety cabinet/Tissue culture (TC) laminar flow hood Chemical hood (for ethane and propane) Low-speed centrifuge (Eppendorf 5702 or equivalent) Hemocytometer or equivalent cell counter Inverted microscope for cell culture (Olympus or equivalent) Cell culture dishes 10cm, tissue culture treated (Corning, #CLS430599) 35 mm MatTek culture dishes, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated (MatTek, #P35G-1.5-14) Pipettes and pipette tips Parafilm (VWR, #3-374-5) Vitrobot Mark IV System (FEI/Thermo Fisher Scientific) and Vitrobot accessories ( i.e ., ethane tray, anticontamination ring, Vitrobot tweezers, holder for grid boxes, cooling spider, brass ethane cup) Whatman filter paper (GE Healthcare Life Sciences, #1001-055 and #1440-150) 60 mL syringe (Cole-Parmer, #0794542) Tweezers for EM grid handling (Dumont, #0203-L5-P0) Large tweezers for handling EM grid boxes (VWR, #82027-432) UltrAuFoil ® Gold R 2/2 on Gold 200 EM grids (Quantifoil/SPT Labtech, # N1-A16nAu20-01; alternative option: Electron Microscopy Sciences, #LFH2100AR2I) or alternatively QUANTIFOIL ® Carbon R 2/2 on Gold 200 (Quantifoil/SPT Labtech, #N1-C16nAu20-01; alternative option: Electron Microscopy Sciences, #Q2100AR2) Cryo grid box, round, with lid (Electron Microscopy Sciences, #71166-10-W) Screwdriver kit (Ted Pella, #53719) 350 mL Dewar shielded vacuum flask (Pope Scientific, #8600/0099) Liquid nitrogen (LN 2 ) storage unit for storing frozen samples ( e.g ., Thermo Fisher Scientific Locator cryogenic storage system) 15 mL and 50 mL conical tubes (Fisher Scientific, #14955237 and #1495949A) Kimwipes String, yard (to pull the samples out the liquid nitrogen) Liquid nitrogen (Matheson, #NIL160-22) Ethane (Matheson, #UN1035) Propane (Matheson, #UN1978) ..
Electron Microscopy:Article Title: Preparation of vitrified mammalian cells for in situ cryo-electron tomography
Article Snippet: .. INS-1E cells (gift of P. Maechler, Université de Genève) ( Merglen et al., 2004 ) Dulbecco's Modified Eagle Medium (DMEM) (Corning, #MT10013CV) Gibco RPMI 1640 Medium (Fisher Scientific, #1875119) Fetal bovine serum (FBS) (Corning, #35-010-CV) HEPES (1M) (Fisher Scientific Company, #15-630-080) Penicillin-streptomycin (10,000 U/mL) (Fisher Scientific Company, #15140122) Sodium pyruvate (100 mM) (Fisher Scientific, #11360070) L-Glutamine (200 mM) (Fisher Scientific, #25030081) 2-mercaptoethanol (Fisher Scientific, #21985023) Dulbecco’s Phosphate-Buffered Saline, 1X without calcium and magnesium (DPBS) 1x (Corning, # 21-031-CM) Trypsin-EDTA 1x (Corning, # 25-052-CI) Human fibronectin (Sigma, #F1056) Trypan Blue Solution, 0.4% (Fisher Scientific, #15250061) 70% Ethanol Mili-Q ddH20 CO 2 incubator Class II biosafety cabinet/Tissue culture (TC) laminar flow hood Chemical hood (for ethane and propane) Low-speed centrifuge (Eppendorf 5702 or equivalent) Hemocytometer or equivalent cell counter Inverted microscope for cell culture (Olympus or equivalent) Cell culture dishes 10cm, tissue culture treated (Corning, #CLS430599) 35 mm MatTek culture dishes, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated (MatTek, #P35G-1.5-14) Pipettes and pipette tips Parafilm (VWR, #3-374-5) Vitrobot Mark IV System (FEI/Thermo Fisher Scientific) and Vitrobot accessories ( i.e ., ethane tray, anticontamination ring, Vitrobot tweezers, holder for grid boxes, cooling spider, brass ethane cup) Whatman filter paper (GE Healthcare Life Sciences, #1001-055 and #1440-150) 60 mL syringe (Cole-Parmer, #0794542) Tweezers for EM grid handling (Dumont, #0203-L5-P0) Large tweezers for handling EM grid boxes (VWR, #82027-432) UltrAuFoil ® Gold R 2/2 on Gold 200 EM grids (Quantifoil/SPT Labtech, # N1-A16nAu20-01; alternative option: Electron Microscopy Sciences, #LFH2100AR2I) or alternatively QUANTIFOIL ® Carbon R 2/2 on Gold 200 (Quantifoil/SPT Labtech, #N1-C16nAu20-01; alternative option: Electron Microscopy Sciences, #Q2100AR2) Cryo grid box, round, with lid (Electron Microscopy Sciences, #71166-10-W) Screwdriver kit (Ted Pella, #53719) 350 mL Dewar shielded vacuum flask (Pope Scientific, #8600/0099) Liquid nitrogen (LN 2 ) storage unit for storing frozen samples ( e.g ., Thermo Fisher Scientific Locator cryogenic storage system) 15 mL and 50 mL conical tubes (Fisher Scientific, #14955237 and #1495949A) Kimwipes String, yard (to pull the samples out the liquid nitrogen) Liquid nitrogen (Matheson, #NIL160-22) Ethane (Matheson, #UN1035) Propane (Matheson, #UN1978) ..
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