Plasmid Preparation:Article Title: Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
Article Snippet: Materials 70% ethanol Human plasma fibronectin diluted in 1 × PBS (without divalent cations) 1% (w/v) BSA/DMEM HG, heat-inactivated (see recipe) HFF-1 growth medium (see recipe) 0.05% (w/v) trypsin-0.53mM EDTA Cell Line 96-well Nucleofector Kit SE (Amaxa, VHCA-1001) containing: SE solution Supplement Plasmid: tdEos/paxillin (~0.5 to 1 μg/μl) Normal human foreskin fibroblast (HFF-1) cells (ATCC cat. no. SCRC-1041) grown in 75-cm 2 cell culture flasks with 0.2-μm vent caps for 2 to 3 days before transfection Compressed butane/natural gas, burner, and lighter Fine steel forceps Cleaned coverslips (see Support Protocol 1) 35 × 10–mm cell culture dishes 37°C water bath 1.5-ml microcentrifuge tube or 15-ml conical centrifuge tubes Centrifuge capable of 90 × g (Eppendorf 5810 or equivalent) Centrifuge rotor (Eppendorf 5810 A-4-62 or equivalent) Rainin LTS pipets (0.1 to 200 μl) Rainin LTS tips (RT-L10F and RT-L200F) Nucleofector 96-well shuttle system (Amaxa Biosystems) Additional reagents and equipment for performing a viable cell count ( UNIT 1.1 ) Pre-coat coverslips with 10 μg/ml fibronectin (day 1) 1 Spray forceps with 70% ethanol and pass through flame of ignited butane gas to sterilize.
Article Title: Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
Article Snippet: 70% ethanol Human plasma fibronectin diluted in 1 × PBS (without divalent cations) 1% (w/v) BSA/DMEM HG, heat-inactivated (see recipe) HFF-1 growth medium (see recipe) 0.05% (w/v) trypsin-0.53mM EDTA Cell Line 96-well Nucleofector Kit SE (Amaxa, VHCA-1001) containing: SE solution Supplement Plasmid: tdEos/paxillin (~0.5 to 1 μg/μl) Normal human foreskin fibroblast (HFF-1) cells (ATCC cat. no. SCRC-1041) grown in 75-cm 2 cell culture flasks with 0.2-μm vent caps for 2 to 3 days before transfection Compressed butane/natural gas, burner, and lighter Fine steel forceps Cleaned coverslips (see Support Protocol 1) 35 × 10–mm cell culture dishes 37°C water bath 1.5-ml microcentrifuge tube or 15-ml conical centrifuge tubes Centrifuge capable of 90 × g (Eppendorf 5810 or equivalent) Centrifuge rotor (Eppendorf 5810 A-4-62 or equivalent) Rainin LTS pipets (0.1 to 200 μl) Rainin LTS tips (RT-L10F and RT-L200F) Nucleofector 96-well shuttle system (Amaxa Biosystems) Additional reagents and equipment for performing a viable cell count ( UNIT 1.1 )
Cell Culture:Article Title: Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
Article Snippet: Materials 70% ethanol Human plasma fibronectin diluted in 1 × PBS (without divalent cations) 1% (w/v) BSA/DMEM HG, heat-inactivated (see recipe) HFF-1 growth medium (see recipe) 0.05% (w/v) trypsin-0.53mM EDTA Cell Line 96-well Nucleofector Kit SE (Amaxa, VHCA-1001) containing: SE solution Supplement Plasmid: tdEos/paxillin (~0.5 to 1 μg/μl) Normal human foreskin fibroblast (HFF-1) cells (ATCC cat. no. SCRC-1041) grown in 75-cm 2 cell culture flasks with 0.2-μm vent caps for 2 to 3 days before transfection Compressed butane/natural gas, burner, and lighter Fine steel forceps Cleaned coverslips (see Support Protocol 1) 35 × 10–mm cell culture dishes 37°C water bath 1.5-ml microcentrifuge tube or 15-ml conical centrifuge tubes Centrifuge capable of 90 × g (Eppendorf 5810 or equivalent) Centrifuge rotor (Eppendorf 5810 A-4-62 or equivalent) Rainin LTS pipets (0.1 to 200 μl) Rainin LTS tips (RT-L10F and RT-L200F) Nucleofector 96-well shuttle system (Amaxa Biosystems) Additional reagents and equipment for performing a viable cell count ( UNIT 1.1 ) Pre-coat coverslips with 10 μg/ml fibronectin (day 1) 1 Spray forceps with 70% ethanol and pass through flame of ignited butane gas to sterilize.
Article Title: Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
Article Snippet: 70% ethanol Human plasma fibronectin diluted in 1 × PBS (without divalent cations) 1% (w/v) BSA/DMEM HG, heat-inactivated (see recipe) HFF-1 growth medium (see recipe) 0.05% (w/v) trypsin-0.53mM EDTA Cell Line 96-well Nucleofector Kit SE (Amaxa, VHCA-1001) containing: SE solution Supplement Plasmid: tdEos/paxillin (~0.5 to 1 μg/μl) Normal human foreskin fibroblast (HFF-1) cells (ATCC cat. no. SCRC-1041) grown in 75-cm 2 cell culture flasks with 0.2-μm vent caps for 2 to 3 days before transfection Compressed butane/natural gas, burner, and lighter Fine steel forceps Cleaned coverslips (see Support Protocol 1) 35 × 10–mm cell culture dishes 37°C water bath 1.5-ml microcentrifuge tube or 15-ml conical centrifuge tubes Centrifuge capable of 90 × g (Eppendorf 5810 or equivalent) Centrifuge rotor (Eppendorf 5810 A-4-62 or equivalent) Rainin LTS pipets (0.1 to 200 μl) Rainin LTS tips (RT-L10F and RT-L200F) Nucleofector 96-well shuttle system (Amaxa Biosystems) Additional reagents and equipment for performing a viable cell count ( UNIT 1.1 )
Transfection:Article Title: Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
Article Snippet: Materials 70% ethanol Human plasma fibronectin diluted in 1 × PBS (without divalent cations) 1% (w/v) BSA/DMEM HG, heat-inactivated (see recipe) HFF-1 growth medium (see recipe) 0.05% (w/v) trypsin-0.53mM EDTA Cell Line 96-well Nucleofector Kit SE (Amaxa, VHCA-1001) containing: SE solution Supplement Plasmid: tdEos/paxillin (~0.5 to 1 μg/μl) Normal human foreskin fibroblast (HFF-1) cells (ATCC cat. no. SCRC-1041) grown in 75-cm 2 cell culture flasks with 0.2-μm vent caps for 2 to 3 days before transfection Compressed butane/natural gas, burner, and lighter Fine steel forceps Cleaned coverslips (see Support Protocol 1) 35 × 10–mm cell culture dishes 37°C water bath 1.5-ml microcentrifuge tube or 15-ml conical centrifuge tubes Centrifuge capable of 90 × g (Eppendorf 5810 or equivalent) Centrifuge rotor (Eppendorf 5810 A-4-62 or equivalent) Rainin LTS pipets (0.1 to 200 μl) Rainin LTS tips (RT-L10F and RT-L200F) Nucleofector 96-well shuttle system (Amaxa Biosystems) Additional reagents and equipment for performing a viable cell count ( UNIT 1.1 ) Pre-coat coverslips with 10 μg/ml fibronectin (day 1) 1 Spray forceps with 70% ethanol and pass through flame of ignited butane gas to sterilize.
Article Title: Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
Article Snippet: 70% ethanol Human plasma fibronectin diluted in 1 × PBS (without divalent cations) 1% (w/v) BSA/DMEM HG, heat-inactivated (see recipe) HFF-1 growth medium (see recipe) 0.05% (w/v) trypsin-0.53mM EDTA Cell Line 96-well Nucleofector Kit SE (Amaxa, VHCA-1001) containing: SE solution Supplement Plasmid: tdEos/paxillin (~0.5 to 1 μg/μl) Normal human foreskin fibroblast (HFF-1) cells (ATCC cat. no. SCRC-1041) grown in 75-cm 2 cell culture flasks with 0.2-μm vent caps for 2 to 3 days before transfection Compressed butane/natural gas, burner, and lighter Fine steel forceps Cleaned coverslips (see Support Protocol 1) 35 × 10–mm cell culture dishes 37°C water bath 1.5-ml microcentrifuge tube or 15-ml conical centrifuge tubes Centrifuge capable of 90 × g (Eppendorf 5810 or equivalent) Centrifuge rotor (Eppendorf 5810 A-4-62 or equivalent) Rainin LTS pipets (0.1 to 200 μl) Rainin LTS tips (RT-L10F and RT-L200F) Nucleofector 96-well shuttle system (Amaxa Biosystems) Additional reagents and equipment for performing a viable cell count ( UNIT 1.1 )
Cell Counting:Article Title: Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
Article Snippet: Materials 70% ethanol Human plasma fibronectin diluted in 1 × PBS (without divalent cations) 1% (w/v) BSA/DMEM HG, heat-inactivated (see recipe) HFF-1 growth medium (see recipe) 0.05% (w/v) trypsin-0.53mM EDTA Cell Line 96-well Nucleofector Kit SE (Amaxa, VHCA-1001) containing: SE solution Supplement Plasmid: tdEos/paxillin (~0.5 to 1 μg/μl) Normal human foreskin fibroblast (HFF-1) cells (ATCC cat. no. SCRC-1041) grown in 75-cm 2 cell culture flasks with 0.2-μm vent caps for 2 to 3 days before transfection Compressed butane/natural gas, burner, and lighter Fine steel forceps Cleaned coverslips (see Support Protocol 1) 35 × 10–mm cell culture dishes 37°C water bath 1.5-ml microcentrifuge tube or 15-ml conical centrifuge tubes Centrifuge capable of 90 × g (Eppendorf 5810 or equivalent) Centrifuge rotor (Eppendorf 5810 A-4-62 or equivalent) Rainin LTS pipets (0.1 to 200 μl) Rainin LTS tips (RT-L10F and RT-L200F) Nucleofector 96-well shuttle system (Amaxa Biosystems) Additional reagents and equipment for performing a viable cell count ( UNIT 1.1 ) Pre-coat coverslips with 10 μg/ml fibronectin (day 1) 1 Spray forceps with 70% ethanol and pass through flame of ignited butane gas to sterilize.
Article Title: Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
Article Snippet: 70% ethanol Human plasma fibronectin diluted in 1 × PBS (without divalent cations) 1% (w/v) BSA/DMEM HG, heat-inactivated (see recipe) HFF-1 growth medium (see recipe) 0.05% (w/v) trypsin-0.53mM EDTA Cell Line 96-well Nucleofector Kit SE (Amaxa, VHCA-1001) containing: SE solution Supplement Plasmid: tdEos/paxillin (~0.5 to 1 μg/μl) Normal human foreskin fibroblast (HFF-1) cells (ATCC cat. no. SCRC-1041) grown in 75-cm 2 cell culture flasks with 0.2-μm vent caps for 2 to 3 days before transfection Compressed butane/natural gas, burner, and lighter Fine steel forceps Cleaned coverslips (see Support Protocol 1) 35 × 10–mm cell culture dishes 37°C water bath 1.5-ml microcentrifuge tube or 15-ml conical centrifuge tubes Centrifuge capable of 90 × g (Eppendorf 5810 or equivalent) Centrifuge rotor (Eppendorf 5810 A-4-62 or equivalent) Rainin LTS pipets (0.1 to 200 μl) Rainin LTS tips (RT-L10F and RT-L200F) Nucleofector 96-well shuttle system (Amaxa Biosystems) Additional reagents and equipment for performing a viable cell count ( UNIT 1.1 )
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