3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

1. DNA probe preparation and labelling procedures with nick translation

1. Purify and clean bacterial artificial chromosomes (BACs), plasmids or PCR products with specific kits (Table of Materials), resuspend in ddH₂O, check by electrophoresis on an agarose gel and quantify.
2. Perform nick translation on 1.5−2 µg of DNA from step 1.1 in a final volume of 50 µL by mixing all the reagents in a 0.5 mL low binding DNA tube according to Table 1.
   NOTE: Directly labelled probes can improve the signal to noise ratio. Kits to produce indirectly and directly labelled probes with various Alexa fluorochromes by nick translation are commercially available.
3. Incubate the nick translation mix in a thermal mixer at 16 °C for a time depending on the length of the starting DNA material: 45 min for PCR products (a pool of PCR products of 2,000 bp each) and up to 4 h for BAC DNA and plasmids.
4. Check the size of the probes produced in step 1.3 by electrophoresis on a 2.2% agarose gel.
   NOTE: The optimal probe size is <200 bp (Figure 1A).
   1. If the DNA is not digested enough, add 5 U of DNA polymerase I and 0.05 U of DNase I to the reaction and incubate for 1−2 h at 16 °C and subsequently re-check. Stop the reaction with 0.5 mM EDTA (final concentration). Store probes at -20 °C.
5. For each DNA FISH experiment, precipitate the following quantities of probes depending on the starting DNA material from which the probes are produced: 200 ng from nick translated PCR products, 100 ng from nick translated BACs, or 300 ng from nick translated plasmid. Add ddH₂O up to 150 µL, 20 µg of unlabelled salmon sperm DNA, 3.5 µg of species-specific Cot-1 DNA, 3 volumes of 100% EtOH, and 1/10 volume of 3 M sodium acetate pH 5.2. Precipitate at -80 °C for 1 h.
6. Centrifuge at maximum speed for 1 h at 4 °C and discard the supernatant. Wash the pellet twice with 70% EtOH.
7. Resuspend the pellet in 2 µL of 100% formamide pH 7.0, shake at 40 °C for 30 min (can take up to a few hours) and then add an equal volume of 4x saline-sodium citrate (SSC)/20% dextran sulfate.
   1. Prepare 20x SSC by mixing 175.3 g of NaCl and 88.2 g of sodium citrate in a final volume of 1 L of H₂O. Autoclave, filter and prepare aliquots.
      NOTE: For multicolor DNA FISH, the different probes can be precipitated together except for probes that can anneal with one another. In these specific cases, treat them separately, precipitate and resuspend the probes dividing the reagents mentioned in steps 1.5−1.7 with respect to the number of probes. Pool the probes only after resuspension in formamide. If glass coverslips greater than 10 mm or less than 10 mm are used, scale up/down the volume of the reagents used in steps 1.5−1.7 proportionally to the slide size. Hybridization probes can be stored at -20 °C for a long period of time (up to two months).

2. Cell fixation, pre-treatment and permeabilization

NOTE: Permeabilization and deproteinization passages are crucial steps. The time of reaction and concentration of the reagents strongly depend on the cell type, the cytoplasm abundance, and the nuclear morphology.

1. Cell fixation, pre-treatment and permeabilization for human primary T lymphocytes
   NOTE: This protocol is suitable for small cells, with small nuclei and a low amount of cytoplasm.
   1. Use glass coverslips (10 mm, thickness No. 1.5H).
   2. Wash the glass with ddH₂O, then with 70% EtOH and let them dry. Use one glass for each well of a 24-well plate.
   3. Add 200 µL of 0.1% poly-L-lysine (w/v) (Table of Materials) directly on the glass to form a drop. Pay attention as the drop must remain on the glass surface without touching the well for 2−5 min. Leave out the drop carefully, and let the glass dry for 30 min.
   4. Perform step 2.1.3 twice more.
   5. Put 200 µL of suspension cells (2 x 10⁶/mL, PBS suspension of ex vivo primary T lymphocytes) directly on the glass, allow the cells to seed at room temperature (RT) for 30 min.
   6. Quickly remove the drop. Add freshly made 4% PFA (prepared in PBS/0.1% TWEEN 20, pH 7.0, filtered) for 10 min and fix the seeded cells at RT.
   7. Wash three times for 5 min each with 0.05% Triton X-100/PBS (TPBS) at RT. Permeabilize with 0.5% TPBS for 10 min at RT.
   8. Perform RNase treatment by adding 2.5 µL of RNase cocktail (Table of Materials) in 250 µL of PBS/well in a 24-well plate for 1 h at 37 °C.
   9. Rinse in PBS, add 20% glycerol/PBS, and incubate overnight (ON) at 4 °C.
      NOTE: This step can range from 1 h to ON. Slides can be kept in 20% glycerol/PBS at 4 °C up to 7 days.
   10. Freeze on dry ice (15−30 s), thaw gradually at RT, and soak in 20% glycerol/PBS. Repeat step 2.1.9 another three times.
   11. Wash in 0.5% TPBS for 5 min at RT. Wash in 0.05% TPBS, twice for 5 min at RT. Incubate in 0.1 N HCl for 12 min at RT. Rinse in 2x SSC.
   12. Incubate in 50% formamide pH 7.0/2x SSC ON at RT.
       NOTE: The incubation time can be optimized and eventually reduced. Slides can be kept in 50% formamide/2x SSC for several days.
2. Cell fixation, pre-treatment and permeabilization for human primary myoblasts
   NOTE: This protocol is suitable for large cells, with a high amount of cytoplasm.
   1. Grow human primary myoblasts directly on the coverslip glasses in 24-well plates in growth medium (Dulbecco's modified Eagle medium (DMEM), 20% fetal bovine serum (FBS), 25 ng/mL fibroblast growth factor (FGF), 10 ng/mL epidermal growth factor (EGF), 10 µg/mL human insulin, 1x glutamine, 1x penicillin/streptomycin) for at least 24 h (reaching 50−70% of confluence).
      NOTE: To facilitate the adhesion, gelatin or collagen or poly-L-lysine can be used to coat the coverslip prior the seeding.
   2. Rinse cells in 2−3 changes of PBS. Add freshly made 4% PFA and fix the cells for 10 min at RT.
   3. Wash three times for 3 min each in 0.01% TPBS at RT. Permeabilize in 0.5% TPBS for 10 min at RT.
   4. Perform RNase treatment by adding 2.5 µL of RNase cocktail (Table of Materials) in 250 µL of PBS/well in a 24-well plate for 1 h at 37 °C.
   5. Rinse in PBS, add 20% glycerol/PBS, and incubate ON at RT.
      NOTE: This step can range from 1 h to ON. Slides can be kept in 20% glycerol/PBS at 4 °C up to 7 days.
   6. Freeze on dry ice (15−30 s), thaw gradually at RT, and soak in 20% glycerol/PBS. Repeat this step three times.
   7. Wash three times for 10 min each in PBS. Incubate in 0.1 N HCl for 5 min at RT. Rinse in 2x SSC.
   8. Incubate in 50% formamide pH 7.0/2x SSC ON at RT.
      NOTE: The time of incubation can be optimized and eventually reduced. Slides can be kept in 50% formamide/2x SSC for several days.
   9. Equilibrate slides (kept in 50% formamide/2x SSC) in 2x SSC for 2 min. Then, equilibrate in PBS for 3 min.
   10. Treat with 0.01 N HCl/0.0025% pepsin from a few seconds up to 5 min, depending on the cell type. During this step, observe the cells under an optical microscope and stop the reaction (step 2.2.11) as soon as the nuclei are free from the cytoplasm, while maintaining their structure intact (e.g., nucleoli are still visible and intact).
   11. Inactivate the pepsin by washing twice for 5 min each in 50 mM MgCl₂/PBS.
   12. Post-fix in 1% PFA/PBS for 1 min. Wash for 5 min in PBS. Wash twice for 5 min each in 2x SSC, and then add 50% formamide/2x SSC for at least 30 min.

3. 3D multicolor DNA FISH hybridization

1. Denature the probes at 80 °C for 5 min, and then put quickly on ice.
2. Load the hybridization probes on a clean microscope slide. Turn the coverslip with cells upside down on the drop of hybridization probes.
3. Seal the coverslip with rubber cement. Let the rubber cement dry completely. Then place slides on a heating block and denature at 75 °C for 4 min.
   NOTE: The timing of denaturation and temperature of denaturation can be augmented, up to 80 °C.
4. Hybridize at 37 °C ON in a metallic box floating in a water bath.
   NOTE: To improve the signal to noise ratio, the hybridization temperature can go up to 42 °C.
5. Peel off rubber cement, immerse the slides in 2x SCC, strip off the glass coverslip and transfer it to 2x SSC in 6-well plate.
6. Wash in 2x SSC three times for 5 min each at 37 °C, shaking at 90 rpm in an incubator shaker. Wash in 0.1x SSC three times for 5 min each at 60 °C, shaking at 90 rpm in an incubator shaker. Rinse briefly in 4x SSC/0.2% TWEEN 20.

4. 3D multicolor DNA FISH detection

NOTE: For directly labelled probes, skip steps 4.1 and 4.2.

1. Block in 4x SSC/0.2% TWEEN 20/4% bovine serum albumin (BSA) for 20 min at 37 °C in a 24-well plate, shaking at 20 rpm in an incubator shaker.
2. Incubate with the appropriate concentration of anti-digoxygenin (1:150), and/or streptavidin (1:1,000) (Table of Materials) diluted in 4x SSC/0.2% TWEEN 20/4% BSA for 35 min in a dark and wet chamber at 37 °C.
3. Wash in 4x SSC/0.2% TWEEN 20 three times for 5 min each at 37 °C, shaking at 90 rpm in a 6-well plate in an incubator shaker.
4. Equilibrate in PBS and post-fix in 2% formaldehyde/PBS for 2 min at RT in a 24-well plate.
   NOTE: Directly labelled probes do not need post-fixation.
5. Wash 5x briefly in PBS. Stain with 1 ng/mL DAPI (4,6-diamidino-2-phenylindole)/PBS for 5 min at RT.
6. Wash 5x briefly in PBS. Mount with antifade solution (Table of Materials).

5. 3D multicolor DNA FISH microscopy and analysis

1. Acquire 3D images with a microscope system.
   NOTE: Here, a widefield microscope with an axial distance of 0.2−0.25 μm between consecutive sections (Figure 1B) is used.
2. Analyze 3D image stacks using different software and tools (here, NuCLεD or Nuclear Contacts Locator in 3D).
   NOTE: The tool NuCLεD has been developed in order to automatically analyze 3D multicolor DNA FISH in fluorescence cell image z-stacks. NuCLεD reconstructs the nuclei from cell image stacks in 3D as well as detects and localizes fluorescent 3D spots. It measures the relative positioning of spots in the nucleus (e.g., distance from the centroid of the nuclei and/or periphery of the nuclei) and the maximum radius for each nucleus and inter-spots distances. The tool and the algorithm used are fully described in Cortesi et al.¹⁶.
3. Analyze the data (e.g., 3D distances between specified genomic loci and the nuclear centroid and contact frequencies) retrieved by NuCLεD.
   NOTE: For 3D distances between specified genomic loci and the nuclear centroid, normalize distances on the maximum radius for each nucleus and represent these data as frequency distributions of normalized distances from the nuclear centroid. For long range interactions studies, contact frequencies are supposed to be in the range of 10−20%²¹ with a threshold inter-distance that can vary and can be put around 2 µm³⁵.
   1. To perform statistical analysis, analyze approximately 100 nuclei per biological replicate. Represent 3D distances between specified genomic loci as cumulative frequency distributions of distances that are below the threshold inter-distance selected and use a t-test to assess the significance of differences in the distributions. Also, calculate the percentage of nuclei positive for the interactions that are below the threshold inter-distance selected, and use Fisher's exact test to assess the significance of differences in the percentages.