8. mTFP1-TRAF-Venus: low FRET standard construct made of
mTFP1 and Venus separated by a 229 amino acid from Tumor
Necrosis Factor Receptor Associated Factor domain (referred
to as TRAF) [17].
All cell lines and constructs are available on demand.
2.2 Cell Imaging
1. Any commercial #1.5 glass-bottomed imaging dish.
2. Collagen from human placenta Bornstein and Traub Type IV
(referred to as collagen) 50 μg/mL in acetic acid solution:
solubilize 5 mg of collagen in 5 mL of acetic acid (0.5 M) on
ice to obtain a 20Â stock solution. Dilute in acetic acid (0.5 M)
to obtain a solution at the final concentration of 50 μg/mL.
3. Imaging medium: FluoroBrite™ DMEM (or any equivalent
product), without phenol red, supplemented with 10% v/v
FBS, 1% v/v PS, 2.5 mM L-Glutamine and 20 mM HEPES.
Store away from light.
4. Starvation medium: FluoroBrite™ DMEM, without phenol
red, supplemented with 0.5% v/v FBS, 1% v/v PS, 2.5 mM LGlutamine and 20 mM HEPES. Store away from light.
5. Paraffin oil. Store away from the light at room temperature.
2.3 Cell
Scatter Assay
1. Hepatocyte Growth Factor (HGF) 10 ng/μL in BSA/DPBS
solution: solubilize 1 mg of BSA (Bovine Serum Albumin) in
1 mL of DPBS. Solubilize 10 μg of HGF in the BSA/DPBS
solution. Stock as 10 μL aliquots.
2. Stimulation medium: The day of the experiment, dilute an
aliquot in 2 mL of starvation medium for a final HGF concentration of 50 ng/mL.
2.4 Wound
Healing Assay
1. 200 μL pipette tips.
2. Washing medium.
2.5 Microscope
1. Commercial confocal inverted microscope equipped with an
incubator (37
C and 5% CO 2 in a humidified atmosphere).
2. High numerical aperture immersion objectives (see Note 2).
3. Argon laser—the 458 nm line is optimal for mTFP1 excitation—or equivalent, and a beam splitter that transmits above
the excitation wavelength.
4. Spectral acquisition: microscopes that use a grating in the
emission path (such as Zeiss or Olympus) allow for simultaneous acquisition of the donor and acceptor emission spectra
with a spectral resolution below 10 nm and nm precision (see
Fig. 2b, c).
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Helena Canever et al.
mTFP1 and Venus separated by a 229 amino acid from Tumor
Necrosis Factor Receptor Associated Factor domain (referred
to as TRAF) [17].
All cell lines and constructs are available on demand.
2.2 Cell Imaging
1. Any commercial #1.5 glass-bottomed imaging dish.
2. Collagen from human placenta Bornstein and Traub Type IV
(referred to as collagen) 50 μg/mL in acetic acid solution:
solubilize 5 mg of collagen in 5 mL of acetic acid (0.5 M) on
ice to obtain a 20Â stock solution. Dilute in acetic acid (0.5 M)
to obtain a solution at the final concentration of 50 μg/mL.
3. Imaging medium: FluoroBrite™ DMEM (or any equivalent
product), without phenol red, supplemented with 10% v/v
FBS, 1% v/v PS, 2.5 mM L-Glutamine and 20 mM HEPES.
Store away from light.
4. Starvation medium: FluoroBrite™ DMEM, without phenol
red, supplemented with 0.5% v/v FBS, 1% v/v PS, 2.5 mM LGlutamine and 20 mM HEPES. Store away from light.
5. Paraffin oil. Store away from the light at room temperature.
2.3 Cell
Scatter Assay
1. Hepatocyte Growth Factor (HGF) 10 ng/μL in BSA/DPBS
solution: solubilize 1 mg of BSA (Bovine Serum Albumin) in
1 mL of DPBS. Solubilize 10 μg of HGF in the BSA/DPBS
solution. Stock as 10 μL aliquots.
2. Stimulation medium: The day of the experiment, dilute an
aliquot in 2 mL of starvation medium for a final HGF concentration of 50 ng/mL.
2.4 Wound
Healing Assay
1. 200 μL pipette tips.
2. Washing medium.
2.5 Microscope
1. Commercial confocal inverted microscope equipped with an
incubator (37
C and 5% CO 2 in a humidified atmosphere).
2. High numerical aperture immersion objectives (see Note 2).
3. Argon laser—the 458 nm line is optimal for mTFP1 excitation—or equivalent, and a beam splitter that transmits above
the excitation wavelength.
4. Spectral acquisition: microscopes that use a grating in the
emission path (such as Zeiss or Olympus) allow for simultaneous acquisition of the donor and acceptor emission spectra
with a spectral resolution below 10 nm and nm precision (see
Fig. 2b, c).
292
Helena Canever et al.