Transferring:Article Title: Protocol: High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples
Article Snippet: · 25% (w/v) Polymethylmethacrylate epoxide resin (PMME, Macro-Prep; Bio-Rad, cat. no. 156–0000) suspension in 0.1 M NaHCO 3 (pH 7.0) · 25% (w/v) NH 2 resin (Agilent, cat. no. 12213020) suspension in distilled water · 25% (w/v) DOWEX® 50X2-400 ion-exchange resin (Sigma-Aldrich, cat. no. 217476), H + form, suspension in distilled water · 25% (w/v) Oasis® HLB resin (collected from HLB cartridges; Waters, cat. no. WAT106202) suspension in methanol · Ethereal diazomethane (prepared as previously described [ , ]) · Methyl chloroformate (MCF; Sigma-Aldrich, cat. no. M35304) ► CAUTION : flammable, highly toxic by inhalation, toxic by ingestion, corrosive. .. · MICROMAN positive-displacement pipettes (Gilson, cat. no. F148501, F148502, F148503, F148504, F148505, F148506) · MICROMAN positive-displacement pipette tips (Gilson, cat. no. F148412, F148112, F148113, F148414, F148114, F148560) · 10–200 μl Empty TopTip for solid phase extraction (SPE) and adaptors for centrifugation (Glygen, cat. no. TT2EMT) · Repeater® plus positive displacement pipette (Eppendorf, cat. no. 022260201) · Eppendorf Combitips plus (1.0 ml, 5.0 ml; Eppendorf, cat. no. 022266209, 022266403) · Tungsten-carbide beads (2.38 mm; Craig Ball Sales, cat. no. CATU.002380.000.0010) · SealRite® microcentrifuge tubes (0.5 ml, 2.0 ml; USA Scientific, cat. no. 1605–0000, 1620–2700) · Teflon Mixer-Mill adapter for 1.5- to 2-ml microcentrifuge tubes (Qiagen, cat. no. 69984) · Vibration Mill (Mixer-Mill; Qiagen, cat. no. MM300) · Microcentrifuge (Eppendorf, cat. no. 5417R) · 2-ml Screw capped micro tubes (Sarstedt, cat. no. 72.694) · 2-ml Clear glass vials with polytetrafluoroethylene (PTFE)-lined caps (Fisher Scientific, cat. no. 03-391-7A) · 2-ml Amber glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-8A) · 8-ml Clear glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-7C) · 4-ml Amber glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-8B) · Color pHast Strips (Fisher Scientific, cat. no. S60170) · Wide-mouth crimp vials (Chrom Tech, cat. no. CTV-1104) · 250 μl Glass inserts with bottom spring (Chrom Tech, cat. no. CTI-9425) · Crimp cap with silicone rubber septum, PTFE coated (Chrom Tech, cat. no. 515011) · Crimper for 11-mm crimp caps (Sigma-Aldrich, cat. no. 33195) · Decapping pliers (Chrom Tech, cat. no. 904371) · Micro-dissecting forceps (Sigma-Aldrich, cat. no. F4017) · Gas chromatograph (GC)-mass spectrometer (MS): Trace GC Ultra with TriPlus autosampler, TSQ Quantum triple quadrupole MS (Thermo Scientific®) · GC capillary column 1: HP-5 ms, 30 m, 0.25 mm diameter, 0.25 μm film thickness (Agilent Technologies, cat. no. 19091 S-433UI) · GC capillary column 2 (for analysis of indole-3-pyruvic acid only): DB-17 ms, 30 m, 0.25 mm diameter, 0.25 μm film thickness (Agilent Technologies, cat. no. 122–4732) .. The following items are needed: · Glass diazomethane generator with clear-seal joints as described in Cohen [ ] · Teflon tubing (3 mm, Cole-Parmer, cat. no. C-06407-10) · 8-ml Clear glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-7C · Stainless steel evaporator (six-position evaporator, Barvap 6, Zanntek, cat. no. 11–06000) · Sand bath heated to 55°C The stainless steel evaporator is connected with N 2 gas tank by Teflon tubing, and the probes of the evaporator are positioned above the sand bath so that samples can be heated while being evaporated by flowing N 2 gas.
Article Title: Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging
Article Snippet: ColorpHast pH-indicator-strips with pH range 6.5–10 (Merck, catalog number: 109543) 3. .. 10–100 and 1,000 μl Gilson MICROMAN® positive displacement pipette tips (Gilson, catalog numbers: FD10004, FD10006) 4. .. 10 cm tissue culture dish (Thermo Fisher Scientific, catalog number: 150350) 5.
Article Title: Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging
Article Snippet: Add 200 μl DMSO (Sigma-Aldrich) to dye tube and mix (wrapped in foil) on a rotational mixer for 1 h Use immediately and store excess in 10–20 μl aliquots Store at −80 °C Sodium Chloride (NaCl), 8% solution list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 Dissolve 4 g NaCl (Sigma-Aldrich) in 500 ml distilled water, mix well Filter sterilize using a 0.2 μm filter 50 mM Tris buffer (pH 7.4) list-behavior=simple prefix-word= mark-type=none max-label-size=0 5 ml 1 M Tris (KD Medical) Add distilled water to 100 ml Filter sterilize using a 0.2 μm filter Store up to 1 year at room temperature .. MatTek Dishes (35 mm, #1.5 coverslip, 20 mm opening: MATTEK, catalog number: P35G-1.5-20-C) ColorpHast pH-indicator-strips with pH range 6.5–10 (Merck, catalog number: 109543) 10–100 and 1,000 μl Gilson MICROMAN ® positive displacement pipette tips (Gilson, catalog numbers: FD10004, FD10006) 10 cm tissue culture dish (Thermo Fisher Scientific, catalog number: 150350) Cell lifters (Corning, catalog number: 3008) Aluminum foil Plastic wrap Scintillation vial (Sigma-Aldrich, catalog number: Z190527) 1.5 ml microfuge tubes (Thermo Fisher Scientific, catalog number: AM12400) Dubecco’s Minimal Essential Medium (DMEM) powder, Phenol red (Sigma-Aldrich, catalog number: D2429) NaOH pellets (Sigma-Aldrich, catalog number: S8045) Phosphate Buffered Saline with Calcium and Magnesium (PBS ++ ) chilled to 4 °C and at room temperature (GE Healthcare, Hyclone TM , catalog number: SH30264.02) Rat tail collagen solution (dissolved in 20 mM acetic acid, commercial brands are fine, but in-house preparations are usually cleaner and polymerize faster) at a concentration greater than 5 mg/ml (6 mg/ml used here) Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034) Boric acid (powder 99.5%, Sigma-Aldrich, catalog number: B6768) Acetic acid (Sigma-Aldrich, catalog number: 27221-1L) (chilled to 4 °C, less than 50 ml of 16.65 M is required) Atto-488 NHS-ester 1 mg (Sigma-Aldrich, catalog number: 41698) DMSO (Sigma-Aldrich, catalog number: D2650) Sircol Collagen Assay kit (available from Accurate Chemical & Scientific, catalog number: CLRS1000) Slide-A-Lyzer Dialysis cassette (G2) with 20,000 MW cutoff (Thermo Fisher Scientific, catalog number: 87735) 1 M Tris (pH 7.4, KD Medical, catalog number: RGF-3340) NaCl (Sigma-Aldrich, catalog number: S7653) HCl (37%, Sigma-Aldrich, catalog number: H1758-500ML) 10x DMEM (see Recipes) 10x reconstitution buffer (10x RB; see Recipes) 1 N NaOH (500 μl in a microfuge tube; see Recipes) 1 N HCl (500 μl in a microfuge tube; see Recipes) 50 mM Borate buffer pH 9.0 (see Recipes) 5 mg/ml Atto-488 NHS-ester dye in DMSO (see Recipes) Sodium Chloride (NaCl), 8% solution (see Recipes) 50 mM Tris buffer (see Recipes) .. 10–100 and 1,000 μl Gilson MICROMAN ® positive displacement pipette (Gilson, catalog numbers: F148314, F148180) Lab timer 2–4 L beaker Ultra-clear 8 × 20 mm centrifuge tubes (Optional: Beckman Coulter, catalog number: 345843) Rectangular ice bucket packed with ice Water bath Large Magnetic stir bar to large beaker (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z284491) Small Magnetic stir bar for scintillation vial (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z126942) Stir plate Mini centrifuge ( i.e. , VWR, model: Galaxy Mini) Biological hood (any brand) Brightfield microscope with 10 and 20x phase contrast objectives Cooled centrifuge capable of 20,000 × g and 4 °C ( i.e. , TOMY, model: MX-307) Lab Rocker ( i.e. , Denville Scientific, model: 110, catalog number: S2110) Airfuge ultra centrifuge (optional: Beckman Coulter, catalog number: 340400) Laser scanning or spinning disk confocal microscope ( i.e. , Nikon Instruments, model: A1R or Yokogawa, model: CSU-X1)
Extraction:Article Title: Protocol: High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples
Article Snippet: · 25% (w/v) Polymethylmethacrylate epoxide resin (PMME, Macro-Prep; Bio-Rad, cat. no. 156–0000) suspension in 0.1 M NaHCO 3 (pH 7.0) · 25% (w/v) NH 2 resin (Agilent, cat. no. 12213020) suspension in distilled water · 25% (w/v) DOWEX® 50X2-400 ion-exchange resin (Sigma-Aldrich, cat. no. 217476), H + form, suspension in distilled water · 25% (w/v) Oasis® HLB resin (collected from HLB cartridges; Waters, cat. no. WAT106202) suspension in methanol · Ethereal diazomethane (prepared as previously described [ , ]) · Methyl chloroformate (MCF; Sigma-Aldrich, cat. no. M35304) ► CAUTION : flammable, highly toxic by inhalation, toxic by ingestion, corrosive. .. · MICROMAN positive-displacement pipettes (Gilson, cat. no. F148501, F148502, F148503, F148504, F148505, F148506) · MICROMAN positive-displacement pipette tips (Gilson, cat. no. F148412, F148112, F148113, F148414, F148114, F148560) · 10–200 μl Empty TopTip for solid phase extraction (SPE) and adaptors for centrifugation (Glygen, cat. no. TT2EMT) · Repeater® plus positive displacement pipette (Eppendorf, cat. no. 022260201) · Eppendorf Combitips plus (1.0 ml, 5.0 ml; Eppendorf, cat. no. 022266209, 022266403) · Tungsten-carbide beads (2.38 mm; Craig Ball Sales, cat. no. CATU.002380.000.0010) · SealRite® microcentrifuge tubes (0.5 ml, 2.0 ml; USA Scientific, cat. no. 1605–0000, 1620–2700) · Teflon Mixer-Mill adapter for 1.5- to 2-ml microcentrifuge tubes (Qiagen, cat. no. 69984) · Vibration Mill (Mixer-Mill; Qiagen, cat. no. MM300) · Microcentrifuge (Eppendorf, cat. no. 5417R) · 2-ml Screw capped micro tubes (Sarstedt, cat. no. 72.694) · 2-ml Clear glass vials with polytetrafluoroethylene (PTFE)-lined caps (Fisher Scientific, cat. no. 03-391-7A) · 2-ml Amber glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-8A) · 8-ml Clear glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-7C) · 4-ml Amber glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-8B) · Color pHast Strips (Fisher Scientific, cat. no. S60170) · Wide-mouth crimp vials (Chrom Tech, cat. no. CTV-1104) · 250 μl Glass inserts with bottom spring (Chrom Tech, cat. no. CTI-9425) · Crimp cap with silicone rubber septum, PTFE coated (Chrom Tech, cat. no. 515011) · Crimper for 11-mm crimp caps (Sigma-Aldrich, cat. no. 33195) · Decapping pliers (Chrom Tech, cat. no. 904371) · Micro-dissecting forceps (Sigma-Aldrich, cat. no. F4017) · Gas chromatograph (GC)-mass spectrometer (MS): Trace GC Ultra with TriPlus autosampler, TSQ Quantum triple quadrupole MS (Thermo Scientific®) · GC capillary column 1: HP-5 ms, 30 m, 0.25 mm diameter, 0.25 μm film thickness (Agilent Technologies, cat. no. 19091 S-433UI) · GC capillary column 2 (for analysis of indole-3-pyruvic acid only): DB-17 ms, 30 m, 0.25 mm diameter, 0.25 μm film thickness (Agilent Technologies, cat. no. 122–4732) .. The following items are needed: · Glass diazomethane generator with clear-seal joints as described in Cohen [ ] · Teflon tubing (3 mm, Cole-Parmer, cat. no. C-06407-10) · 8-ml Clear glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-7C · Stainless steel evaporator (six-position evaporator, Barvap 6, Zanntek, cat. no. 11–06000) · Sand bath heated to 55°C The stainless steel evaporator is connected with N 2 gas tank by Teflon tubing, and the probes of the evaporator are positioned above the sand bath so that samples can be heated while being evaporated by flowing N 2 gas.
Centrifugation:Article Title: Protocol: High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples
Article Snippet: · 25% (w/v) Polymethylmethacrylate epoxide resin (PMME, Macro-Prep; Bio-Rad, cat. no. 156–0000) suspension in 0.1 M NaHCO 3 (pH 7.0) · 25% (w/v) NH 2 resin (Agilent, cat. no. 12213020) suspension in distilled water · 25% (w/v) DOWEX® 50X2-400 ion-exchange resin (Sigma-Aldrich, cat. no. 217476), H + form, suspension in distilled water · 25% (w/v) Oasis® HLB resin (collected from HLB cartridges; Waters, cat. no. WAT106202) suspension in methanol · Ethereal diazomethane (prepared as previously described [ , ]) · Methyl chloroformate (MCF; Sigma-Aldrich, cat. no. M35304) ► CAUTION : flammable, highly toxic by inhalation, toxic by ingestion, corrosive. .. · MICROMAN positive-displacement pipettes (Gilson, cat. no. F148501, F148502, F148503, F148504, F148505, F148506) · MICROMAN positive-displacement pipette tips (Gilson, cat. no. F148412, F148112, F148113, F148414, F148114, F148560) · 10–200 μl Empty TopTip for solid phase extraction (SPE) and adaptors for centrifugation (Glygen, cat. no. TT2EMT) · Repeater® plus positive displacement pipette (Eppendorf, cat. no. 022260201) · Eppendorf Combitips plus (1.0 ml, 5.0 ml; Eppendorf, cat. no. 022266209, 022266403) · Tungsten-carbide beads (2.38 mm; Craig Ball Sales, cat. no. CATU.002380.000.0010) · SealRite® microcentrifuge tubes (0.5 ml, 2.0 ml; USA Scientific, cat. no. 1605–0000, 1620–2700) · Teflon Mixer-Mill adapter for 1.5- to 2-ml microcentrifuge tubes (Qiagen, cat. no. 69984) · Vibration Mill (Mixer-Mill; Qiagen, cat. no. MM300) · Microcentrifuge (Eppendorf, cat. no. 5417R) · 2-ml Screw capped micro tubes (Sarstedt, cat. no. 72.694) · 2-ml Clear glass vials with polytetrafluoroethylene (PTFE)-lined caps (Fisher Scientific, cat. no. 03-391-7A) · 2-ml Amber glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-8A) · 8-ml Clear glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-7C) · 4-ml Amber glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-8B) · Color pHast Strips (Fisher Scientific, cat. no. S60170) · Wide-mouth crimp vials (Chrom Tech, cat. no. CTV-1104) · 250 μl Glass inserts with bottom spring (Chrom Tech, cat. no. CTI-9425) · Crimp cap with silicone rubber septum, PTFE coated (Chrom Tech, cat. no. 515011) · Crimper for 11-mm crimp caps (Sigma-Aldrich, cat. no. 33195) · Decapping pliers (Chrom Tech, cat. no. 904371) · Micro-dissecting forceps (Sigma-Aldrich, cat. no. F4017) · Gas chromatograph (GC)-mass spectrometer (MS): Trace GC Ultra with TriPlus autosampler, TSQ Quantum triple quadrupole MS (Thermo Scientific®) · GC capillary column 1: HP-5 ms, 30 m, 0.25 mm diameter, 0.25 μm film thickness (Agilent Technologies, cat. no. 19091 S-433UI) · GC capillary column 2 (for analysis of indole-3-pyruvic acid only): DB-17 ms, 30 m, 0.25 mm diameter, 0.25 μm film thickness (Agilent Technologies, cat. no. 122–4732) .. The following items are needed: · Glass diazomethane generator with clear-seal joints as described in Cohen [ ] · Teflon tubing (3 mm, Cole-Parmer, cat. no. C-06407-10) · 8-ml Clear glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-7C · Stainless steel evaporator (six-position evaporator, Barvap 6, Zanntek, cat. no. 11–06000) · Sand bath heated to 55°C The stainless steel evaporator is connected with N 2 gas tank by Teflon tubing, and the probes of the evaporator are positioned above the sand bath so that samples can be heated while being evaporated by flowing N 2 gas.
Targeted Proteomics:Article Title: Protocol: High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples
Article Snippet: · 25% (w/v) Polymethylmethacrylate epoxide resin (PMME, Macro-Prep; Bio-Rad, cat. no. 156–0000) suspension in 0.1 M NaHCO 3 (pH 7.0) · 25% (w/v) NH 2 resin (Agilent, cat. no. 12213020) suspension in distilled water · 25% (w/v) DOWEX® 50X2-400 ion-exchange resin (Sigma-Aldrich, cat. no. 217476), H + form, suspension in distilled water · 25% (w/v) Oasis® HLB resin (collected from HLB cartridges; Waters, cat. no. WAT106202) suspension in methanol · Ethereal diazomethane (prepared as previously described [ , ]) · Methyl chloroformate (MCF; Sigma-Aldrich, cat. no. M35304) ► CAUTION : flammable, highly toxic by inhalation, toxic by ingestion, corrosive. .. · MICROMAN positive-displacement pipettes (Gilson, cat. no. F148501, F148502, F148503, F148504, F148505, F148506) · MICROMAN positive-displacement pipette tips (Gilson, cat. no. F148412, F148112, F148113, F148414, F148114, F148560) · 10–200 μl Empty TopTip for solid phase extraction (SPE) and adaptors for centrifugation (Glygen, cat. no. TT2EMT) · Repeater® plus positive displacement pipette (Eppendorf, cat. no. 022260201) · Eppendorf Combitips plus (1.0 ml, 5.0 ml; Eppendorf, cat. no. 022266209, 022266403) · Tungsten-carbide beads (2.38 mm; Craig Ball Sales, cat. no. CATU.002380.000.0010) · SealRite® microcentrifuge tubes (0.5 ml, 2.0 ml; USA Scientific, cat. no. 1605–0000, 1620–2700) · Teflon Mixer-Mill adapter for 1.5- to 2-ml microcentrifuge tubes (Qiagen, cat. no. 69984) · Vibration Mill (Mixer-Mill; Qiagen, cat. no. MM300) · Microcentrifuge (Eppendorf, cat. no. 5417R) · 2-ml Screw capped micro tubes (Sarstedt, cat. no. 72.694) · 2-ml Clear glass vials with polytetrafluoroethylene (PTFE)-lined caps (Fisher Scientific, cat. no. 03-391-7A) · 2-ml Amber glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-8A) · 8-ml Clear glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-7C) · 4-ml Amber glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-8B) · Color pHast Strips (Fisher Scientific, cat. no. S60170) · Wide-mouth crimp vials (Chrom Tech, cat. no. CTV-1104) · 250 μl Glass inserts with bottom spring (Chrom Tech, cat. no. CTI-9425) · Crimp cap with silicone rubber septum, PTFE coated (Chrom Tech, cat. no. 515011) · Crimper for 11-mm crimp caps (Sigma-Aldrich, cat. no. 33195) · Decapping pliers (Chrom Tech, cat. no. 904371) · Micro-dissecting forceps (Sigma-Aldrich, cat. no. F4017) · Gas chromatograph (GC)-mass spectrometer (MS): Trace GC Ultra with TriPlus autosampler, TSQ Quantum triple quadrupole MS (Thermo Scientific®) · GC capillary column 1: HP-5 ms, 30 m, 0.25 mm diameter, 0.25 μm film thickness (Agilent Technologies, cat. no. 19091 S-433UI) · GC capillary column 2 (for analysis of indole-3-pyruvic acid only): DB-17 ms, 30 m, 0.25 mm diameter, 0.25 μm film thickness (Agilent Technologies, cat. no. 122–4732) .. The following items are needed: · Glass diazomethane generator with clear-seal joints as described in Cohen [ ] · Teflon tubing (3 mm, Cole-Parmer, cat. no. C-06407-10) · 8-ml Clear glass vials with PTFE-lined caps (Fisher Scientific, cat. no. 03-391-7C · Stainless steel evaporator (six-position evaporator, Barvap 6, Zanntek, cat. no. 11–06000) · Sand bath heated to 55°C The stainless steel evaporator is connected with N 2 gas tank by Teflon tubing, and the probes of the evaporator are positioned above the sand bath so that samples can be heated while being evaporated by flowing N 2 gas.
Saline:Article Title: Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging
Article Snippet: Add 200 μl DMSO (Sigma-Aldrich) to dye tube and mix (wrapped in foil) on a rotational mixer for 1 h Use immediately and store excess in 10–20 μl aliquots Store at −80 °C Sodium Chloride (NaCl), 8% solution list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 Dissolve 4 g NaCl (Sigma-Aldrich) in 500 ml distilled water, mix well Filter sterilize using a 0.2 μm filter 50 mM Tris buffer (pH 7.4) list-behavior=simple prefix-word= mark-type=none max-label-size=0 5 ml 1 M Tris (KD Medical) Add distilled water to 100 ml Filter sterilize using a 0.2 μm filter Store up to 1 year at room temperature .. MatTek Dishes (35 mm, #1.5 coverslip, 20 mm opening: MATTEK, catalog number: P35G-1.5-20-C) ColorpHast pH-indicator-strips with pH range 6.5–10 (Merck, catalog number: 109543) 10–100 and 1,000 μl Gilson MICROMAN ® positive displacement pipette tips (Gilson, catalog numbers: FD10004, FD10006) 10 cm tissue culture dish (Thermo Fisher Scientific, catalog number: 150350) Cell lifters (Corning, catalog number: 3008) Aluminum foil Plastic wrap Scintillation vial (Sigma-Aldrich, catalog number: Z190527) 1.5 ml microfuge tubes (Thermo Fisher Scientific, catalog number: AM12400) Dubecco’s Minimal Essential Medium (DMEM) powder, Phenol red (Sigma-Aldrich, catalog number: D2429) NaOH pellets (Sigma-Aldrich, catalog number: S8045) Phosphate Buffered Saline with Calcium and Magnesium (PBS ++ ) chilled to 4 °C and at room temperature (GE Healthcare, Hyclone TM , catalog number: SH30264.02) Rat tail collagen solution (dissolved in 20 mM acetic acid, commercial brands are fine, but in-house preparations are usually cleaner and polymerize faster) at a concentration greater than 5 mg/ml (6 mg/ml used here) Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034) Boric acid (powder 99.5%, Sigma-Aldrich, catalog number: B6768) Acetic acid (Sigma-Aldrich, catalog number: 27221-1L) (chilled to 4 °C, less than 50 ml of 16.65 M is required) Atto-488 NHS-ester 1 mg (Sigma-Aldrich, catalog number: 41698) DMSO (Sigma-Aldrich, catalog number: D2650) Sircol Collagen Assay kit (available from Accurate Chemical & Scientific, catalog number: CLRS1000) Slide-A-Lyzer Dialysis cassette (G2) with 20,000 MW cutoff (Thermo Fisher Scientific, catalog number: 87735) 1 M Tris (pH 7.4, KD Medical, catalog number: RGF-3340) NaCl (Sigma-Aldrich, catalog number: S7653) HCl (37%, Sigma-Aldrich, catalog number: H1758-500ML) 10x DMEM (see Recipes) 10x reconstitution buffer (10x RB; see Recipes) 1 N NaOH (500 μl in a microfuge tube; see Recipes) 1 N HCl (500 μl in a microfuge tube; see Recipes) 50 mM Borate buffer pH 9.0 (see Recipes) 5 mg/ml Atto-488 NHS-ester dye in DMSO (see Recipes) Sodium Chloride (NaCl), 8% solution (see Recipes) 50 mM Tris buffer (see Recipes) .. 10–100 and 1,000 μl Gilson MICROMAN ® positive displacement pipette (Gilson, catalog numbers: F148314, F148180) Lab timer 2–4 L beaker Ultra-clear 8 × 20 mm centrifuge tubes (Optional: Beckman Coulter, catalog number: 345843) Rectangular ice bucket packed with ice Water bath Large Magnetic stir bar to large beaker (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z284491) Small Magnetic stir bar for scintillation vial (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z126942) Stir plate Mini centrifuge ( i.e. , VWR, model: Galaxy Mini) Biological hood (any brand) Brightfield microscope with 10 and 20x phase contrast objectives Cooled centrifuge capable of 20,000 × g and 4 °C ( i.e. , TOMY, model: MX-307) Lab Rocker ( i.e. , Denville Scientific, model: 110, catalog number: S2110) Airfuge ultra centrifuge (optional: Beckman Coulter, catalog number: 340400) Laser scanning or spinning disk confocal microscope ( i.e. , Nikon Instruments, model: A1R or Yokogawa, model: CSU-X1)
Concentration Assay:Article Title: Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging
Article Snippet: Add 200 μl DMSO (Sigma-Aldrich) to dye tube and mix (wrapped in foil) on a rotational mixer for 1 h Use immediately and store excess in 10–20 μl aliquots Store at −80 °C Sodium Chloride (NaCl), 8% solution list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 Dissolve 4 g NaCl (Sigma-Aldrich) in 500 ml distilled water, mix well Filter sterilize using a 0.2 μm filter 50 mM Tris buffer (pH 7.4) list-behavior=simple prefix-word= mark-type=none max-label-size=0 5 ml 1 M Tris (KD Medical) Add distilled water to 100 ml Filter sterilize using a 0.2 μm filter Store up to 1 year at room temperature .. MatTek Dishes (35 mm, #1.5 coverslip, 20 mm opening: MATTEK, catalog number: P35G-1.5-20-C) ColorpHast pH-indicator-strips with pH range 6.5–10 (Merck, catalog number: 109543) 10–100 and 1,000 μl Gilson MICROMAN ® positive displacement pipette tips (Gilson, catalog numbers: FD10004, FD10006) 10 cm tissue culture dish (Thermo Fisher Scientific, catalog number: 150350) Cell lifters (Corning, catalog number: 3008) Aluminum foil Plastic wrap Scintillation vial (Sigma-Aldrich, catalog number: Z190527) 1.5 ml microfuge tubes (Thermo Fisher Scientific, catalog number: AM12400) Dubecco’s Minimal Essential Medium (DMEM) powder, Phenol red (Sigma-Aldrich, catalog number: D2429) NaOH pellets (Sigma-Aldrich, catalog number: S8045) Phosphate Buffered Saline with Calcium and Magnesium (PBS ++ ) chilled to 4 °C and at room temperature (GE Healthcare, Hyclone TM , catalog number: SH30264.02) Rat tail collagen solution (dissolved in 20 mM acetic acid, commercial brands are fine, but in-house preparations are usually cleaner and polymerize faster) at a concentration greater than 5 mg/ml (6 mg/ml used here) Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034) Boric acid (powder 99.5%, Sigma-Aldrich, catalog number: B6768) Acetic acid (Sigma-Aldrich, catalog number: 27221-1L) (chilled to 4 °C, less than 50 ml of 16.65 M is required) Atto-488 NHS-ester 1 mg (Sigma-Aldrich, catalog number: 41698) DMSO (Sigma-Aldrich, catalog number: D2650) Sircol Collagen Assay kit (available from Accurate Chemical & Scientific, catalog number: CLRS1000) Slide-A-Lyzer Dialysis cassette (G2) with 20,000 MW cutoff (Thermo Fisher Scientific, catalog number: 87735) 1 M Tris (pH 7.4, KD Medical, catalog number: RGF-3340) NaCl (Sigma-Aldrich, catalog number: S7653) HCl (37%, Sigma-Aldrich, catalog number: H1758-500ML) 10x DMEM (see Recipes) 10x reconstitution buffer (10x RB; see Recipes) 1 N NaOH (500 μl in a microfuge tube; see Recipes) 1 N HCl (500 μl in a microfuge tube; see Recipes) 50 mM Borate buffer pH 9.0 (see Recipes) 5 mg/ml Atto-488 NHS-ester dye in DMSO (see Recipes) Sodium Chloride (NaCl), 8% solution (see Recipes) 50 mM Tris buffer (see Recipes) .. 10–100 and 1,000 μl Gilson MICROMAN ® positive displacement pipette (Gilson, catalog numbers: F148314, F148180) Lab timer 2–4 L beaker Ultra-clear 8 × 20 mm centrifuge tubes (Optional: Beckman Coulter, catalog number: 345843) Rectangular ice bucket packed with ice Water bath Large Magnetic stir bar to large beaker (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z284491) Small Magnetic stir bar for scintillation vial (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z126942) Stir plate Mini centrifuge ( i.e. , VWR, model: Galaxy Mini) Biological hood (any brand) Brightfield microscope with 10 and 20x phase contrast objectives Cooled centrifuge capable of 20,000 × g and 4 °C ( i.e. , TOMY, model: MX-307) Lab Rocker ( i.e. , Denville Scientific, model: 110, catalog number: S2110) Airfuge ultra centrifuge (optional: Beckman Coulter, catalog number: 340400) Laser scanning or spinning disk confocal microscope ( i.e. , Nikon Instruments, model: A1R or Yokogawa, model: CSU-X1)
Sircol Collagen Assay:Article Title: Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging
Article Snippet: Add 200 μl DMSO (Sigma-Aldrich) to dye tube and mix (wrapped in foil) on a rotational mixer for 1 h Use immediately and store excess in 10–20 μl aliquots Store at −80 °C Sodium Chloride (NaCl), 8% solution list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 Dissolve 4 g NaCl (Sigma-Aldrich) in 500 ml distilled water, mix well Filter sterilize using a 0.2 μm filter 50 mM Tris buffer (pH 7.4) list-behavior=simple prefix-word= mark-type=none max-label-size=0 5 ml 1 M Tris (KD Medical) Add distilled water to 100 ml Filter sterilize using a 0.2 μm filter Store up to 1 year at room temperature .. MatTek Dishes (35 mm, #1.5 coverslip, 20 mm opening: MATTEK, catalog number: P35G-1.5-20-C) ColorpHast pH-indicator-strips with pH range 6.5–10 (Merck, catalog number: 109543) 10–100 and 1,000 μl Gilson MICROMAN ® positive displacement pipette tips (Gilson, catalog numbers: FD10004, FD10006) 10 cm tissue culture dish (Thermo Fisher Scientific, catalog number: 150350) Cell lifters (Corning, catalog number: 3008) Aluminum foil Plastic wrap Scintillation vial (Sigma-Aldrich, catalog number: Z190527) 1.5 ml microfuge tubes (Thermo Fisher Scientific, catalog number: AM12400) Dubecco’s Minimal Essential Medium (DMEM) powder, Phenol red (Sigma-Aldrich, catalog number: D2429) NaOH pellets (Sigma-Aldrich, catalog number: S8045) Phosphate Buffered Saline with Calcium and Magnesium (PBS ++ ) chilled to 4 °C and at room temperature (GE Healthcare, Hyclone TM , catalog number: SH30264.02) Rat tail collagen solution (dissolved in 20 mM acetic acid, commercial brands are fine, but in-house preparations are usually cleaner and polymerize faster) at a concentration greater than 5 mg/ml (6 mg/ml used here) Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034) Boric acid (powder 99.5%, Sigma-Aldrich, catalog number: B6768) Acetic acid (Sigma-Aldrich, catalog number: 27221-1L) (chilled to 4 °C, less than 50 ml of 16.65 M is required) Atto-488 NHS-ester 1 mg (Sigma-Aldrich, catalog number: 41698) DMSO (Sigma-Aldrich, catalog number: D2650) Sircol Collagen Assay kit (available from Accurate Chemical & Scientific, catalog number: CLRS1000) Slide-A-Lyzer Dialysis cassette (G2) with 20,000 MW cutoff (Thermo Fisher Scientific, catalog number: 87735) 1 M Tris (pH 7.4, KD Medical, catalog number: RGF-3340) NaCl (Sigma-Aldrich, catalog number: S7653) HCl (37%, Sigma-Aldrich, catalog number: H1758-500ML) 10x DMEM (see Recipes) 10x reconstitution buffer (10x RB; see Recipes) 1 N NaOH (500 μl in a microfuge tube; see Recipes) 1 N HCl (500 μl in a microfuge tube; see Recipes) 50 mM Borate buffer pH 9.0 (see Recipes) 5 mg/ml Atto-488 NHS-ester dye in DMSO (see Recipes) Sodium Chloride (NaCl), 8% solution (see Recipes) 50 mM Tris buffer (see Recipes) .. 10–100 and 1,000 μl Gilson MICROMAN ® positive displacement pipette (Gilson, catalog numbers: F148314, F148180) Lab timer 2–4 L beaker Ultra-clear 8 × 20 mm centrifuge tubes (Optional: Beckman Coulter, catalog number: 345843) Rectangular ice bucket packed with ice Water bath Large Magnetic stir bar to large beaker (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z284491) Small Magnetic stir bar for scintillation vial (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z126942) Stir plate Mini centrifuge ( i.e. , VWR, model: Galaxy Mini) Biological hood (any brand) Brightfield microscope with 10 and 20x phase contrast objectives Cooled centrifuge capable of 20,000 × g and 4 °C ( i.e. , TOMY, model: MX-307) Lab Rocker ( i.e. , Denville Scientific, model: 110, catalog number: S2110) Airfuge ultra centrifuge (optional: Beckman Coulter, catalog number: 340400) Laser scanning or spinning disk confocal microscope ( i.e. , Nikon Instruments, model: A1R or Yokogawa, model: CSU-X1)
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