CDH2, OCLN or VIM, can be custom-prepared using the genomic DNA fragment of interest cloned into BACs and/or fosmids
[15]. DNA-FISH labeled MCF10A cell nuclei are subsequently
imaged in three dimensions, and the loci-nuclear localization analyzed. The complete protocol permits defining relative special
arrangements of loci and correlating them to the physiological
and molecular state of cells undergoing TGF-β induced EMT.
2 Materials
Prepare all solutions using ultrapure water (sensitivity of 18 MΩ cm
at 25
C) and analytical grade reagents. Prepare and store reagents
at room temperature (unless indicated otherwise). Follow all safety
regulations concerning the handling and waste disposal of chemicals (see Note 1).
2.1 Material for Cell
Culture
1. DMEM/F12, containing phenol red, glutamax and 3.1 g/L
D-Glucose.
2. Human epithelial growth factor (hEGF): for stock solution at
7 μg/mL, dissolve 100 μg of commercial hEGF in 14.3 mL
sterile water. Store as 500 μL aliquots at À20
C.
3. 100Â Mammary Epithelial Growth Supplement (MEGS).
4. Cholera toxin: for stock solution at 100 ng/μL, dissolve
0.5 mg of cholera toxin in 5 mL sterile water. 0.2 μm filter
sterilize and store as 500 μL aliquots at À20
C.
5. MCF10A cell line growth medium: for 500 mL DMEM/F12,
add 500 μL of hEGF at 7 μg/mL, 5 mL MEGS (see Note 2),
2.5 mL penicillin-streptomycin at 10,000 U/mL, and 500 μL
of cholera toxin at 100 ng/μL (100 ng/mL final). Store at
4
C.
6. 4 mM HCl with 1 mg/mL BSA: dissolve 250 mg of BSA and
83 μL of 37% HCl in 230 mL of H 2 O. Adjust the volume up to
250 mL. Vortex to dissolve the BSA and filter the solution
using a 0.2 μm filter under a sterile hood. Store at 4
C.
7. TGF-β stock solution at 20 μg/mL: dissolve 2 μg of human
recombinant TGF-β1 in 100 μL of filtrated 4 mM HCl solution
with 1 mg/mL BSA. Store at À20
C.
8. Coverslip #1.5, autoclaved (see Note 3).
9. 1Â Dulbecco’s Phosphate Buffered Saline (DPBS), sterile.
10. 1Â Trypsin—EDTA solution, sterile.
11. Protease inhibitor (Roche) in 1Â PBS: dilute 1 protease inhibitor tablet in 20 mL 1Â PBS.
12. Cell spatula.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
355
[15]. DNA-FISH labeled MCF10A cell nuclei are subsequently
imaged in three dimensions, and the loci-nuclear localization analyzed. The complete protocol permits defining relative special
arrangements of loci and correlating them to the physiological
and molecular state of cells undergoing TGF-β induced EMT.
2 Materials
Prepare all solutions using ultrapure water (sensitivity of 18 MΩ cm
at 25
C) and analytical grade reagents. Prepare and store reagents
at room temperature (unless indicated otherwise). Follow all safety
regulations concerning the handling and waste disposal of chemicals (see Note 1).
2.1 Material for Cell
Culture
1. DMEM/F12, containing phenol red, glutamax and 3.1 g/L
D-Glucose.
2. Human epithelial growth factor (hEGF): for stock solution at
7 μg/mL, dissolve 100 μg of commercial hEGF in 14.3 mL
sterile water. Store as 500 μL aliquots at À20
C.
3. 100Â Mammary Epithelial Growth Supplement (MEGS).
4. Cholera toxin: for stock solution at 100 ng/μL, dissolve
0.5 mg of cholera toxin in 5 mL sterile water. 0.2 μm filter
sterilize and store as 500 μL aliquots at À20
C.
5. MCF10A cell line growth medium: for 500 mL DMEM/F12,
add 500 μL of hEGF at 7 μg/mL, 5 mL MEGS (see Note 2),
2.5 mL penicillin-streptomycin at 10,000 U/mL, and 500 μL
of cholera toxin at 100 ng/μL (100 ng/mL final). Store at
4
C.
6. 4 mM HCl with 1 mg/mL BSA: dissolve 250 mg of BSA and
83 μL of 37% HCl in 230 mL of H 2 O. Adjust the volume up to
250 mL. Vortex to dissolve the BSA and filter the solution
using a 0.2 μm filter under a sterile hood. Store at 4
C.
7. TGF-β stock solution at 20 μg/mL: dissolve 2 μg of human
recombinant TGF-β1 in 100 μL of filtrated 4 mM HCl solution
with 1 mg/mL BSA. Store at À20
C.
8. Coverslip #1.5, autoclaved (see Note 3).
9. 1Â Dulbecco’s Phosphate Buffered Saline (DPBS), sterile.
10. 1Â Trypsin—EDTA solution, sterile.
11. Protease inhibitor (Roche) in 1Â PBS: dilute 1 protease inhibitor tablet in 20 mL 1Â PBS.
12. Cell spatula.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
355