11. 0.1 M HCl. For 50 mL: add 425 μL of 37% HCl to 50 mL
H 2 O.
12. 50% formamide in SSC pH 7–7.2. For 500 mL: combine
250 mL deionized formamide, 50 mL 20Â SSC and 120 mL
H 2 O. Adjust pH with 37% HCl. Store at À20
C.
13. Human Cot-1 DNA at 1 μg/μL.
14. Single-Stranded-DNA (ssDNA) from salmon testes at 10 mg/
mL.
15. Dextran sulfate mix: 20% (w/v) dextran sulfate in 2Â SSC.
Dissolve 20 g of dextran sulfate powder in 80 mL 2Â SSC.
Once the powder is dissolved, adjust the volume by adding
2Â SSC up to 100 mL using a graduated cylinder. Store at
À20
C (see Note 18).
16. Glass slides.
17. Kimwipes.
18. Rubber cement.
19. Heating block.
20. Hoechst 33342, trihydrochloride, trihydrate (10 mg/mL
solution in water): 1/5000 dilution in 1Â PBS. For 10 mL,
dilute 2 μL Hoechst in 10 mL 1Â PBS.
2.5 Material
for Microscopy
Acquisitions
To acquire image stacks we recommend using either a spinning disk
or a conventional confocal microscope, equipped with a 63X HC
PL APO oil immersion objective (NA 1,4–0,7), a Quadrichroic
Mirror (405 nm/488 nm/565 nm/640 nm) for simultaneous
laser excitation, and a SCMOS camera.
Excitation of the aforementioned dyes (Hoechst, Alexa 488,
Alexa 555 and Alexa 647), require lasers with the following wavelengths: 405 nm, 488 nm, 561 nm, and 647 nm.
It is advised to collect fluorescence using single band pass
emission filters adapted to each wavelength.
3 Methods
All steps are carried out at room temperature, unless otherwise
stated.
3.1 Methods for Cell
Culture
Ideally, cells must be cultivated to a maximum of 75–80%
confluency.
3.1.1 MCF10A Cell
Culture
Cultivate MCF10A with the appropriate growing medium (see
Note 19), in 10 mm cell culture dishes for Western Blot and
RT-qPCR analysis and on cleaned coverslips in 6-well plates for
360
Noe ´ mie Kempf et al.
H 2 O.
12. 50% formamide in SSC pH 7–7.2. For 500 mL: combine
250 mL deionized formamide, 50 mL 20Â SSC and 120 mL
H 2 O. Adjust pH with 37% HCl. Store at À20
C.
13. Human Cot-1 DNA at 1 μg/μL.
14. Single-Stranded-DNA (ssDNA) from salmon testes at 10 mg/
mL.
15. Dextran sulfate mix: 20% (w/v) dextran sulfate in 2Â SSC.
Dissolve 20 g of dextran sulfate powder in 80 mL 2Â SSC.
Once the powder is dissolved, adjust the volume by adding
2Â SSC up to 100 mL using a graduated cylinder. Store at
À20
C (see Note 18).
16. Glass slides.
17. Kimwipes.
18. Rubber cement.
19. Heating block.
20. Hoechst 33342, trihydrochloride, trihydrate (10 mg/mL
solution in water): 1/5000 dilution in 1Â PBS. For 10 mL,
dilute 2 μL Hoechst in 10 mL 1Â PBS.
2.5 Material
for Microscopy
Acquisitions
To acquire image stacks we recommend using either a spinning disk
or a conventional confocal microscope, equipped with a 63X HC
PL APO oil immersion objective (NA 1,4–0,7), a Quadrichroic
Mirror (405 nm/488 nm/565 nm/640 nm) for simultaneous
laser excitation, and a SCMOS camera.
Excitation of the aforementioned dyes (Hoechst, Alexa 488,
Alexa 555 and Alexa 647), require lasers with the following wavelengths: 405 nm, 488 nm, 561 nm, and 647 nm.
It is advised to collect fluorescence using single band pass
emission filters adapted to each wavelength.
3 Methods
All steps are carried out at room temperature, unless otherwise
stated.
3.1 Methods for Cell
Culture
Ideally, cells must be cultivated to a maximum of 75–80%
confluency.
3.1.1 MCF10A Cell
Culture
Cultivate MCF10A with the appropriate growing medium (see
Note 19), in 10 mm cell culture dishes for Western Blot and
RT-qPCR analysis and on cleaned coverslips in 6-well plates for
360
Noe ´ mie Kempf et al.