Materials and reagents

Note: Caution must be exercised when performing TASQs staining experiments; the most important features are gathered in the different notes below (see General notes 1–4), which must be read before preparing the materials needed for the experiments. All solutions must be prepared (unless otherwise stated) in ultrapure water (resistivity ≥ 18 MΩ·cm at 25 °C), and molecular biology and/or analytical grade reagents must be used. Solutions can be prepared and stored at room temperature (unless otherwise stated); some of them need to be prepared under a sterile environment in the biological safety cabinet (BSC) and/or to be filtered (PES 0.22 μm bottle-top vacuum filter system or syringe filter system, depending on volume).

Biological materials

1. HeLa cell line (ATCC, catalog number: CCL-2)

Reagents

Note: As not all reagents and/or solutions are absolutely required (see General notes 1–4) and some are mutually exclusive, we strongly recommend reading the procedure in its entirety to determine if they should be used.

Note: We provide the list of reagents we used, but reagents from alternative suppliers are acceptable as long as they are molecular biology and/or analytical-grade reagents.

1. Dulbecco’s modified Eagle medium (DMEM) containing sodium pyruvate, high glucose, and GlutaMAX (Gibco, catalog number: 31966021); store at 4 °C

2. Fetal bovine serum (FBS) 100% (Dutscher, catalog number: on request); store at -80 °C

3. Penicillin/streptomycin 10,000 U/mL (Gibco, catalog number: 15140122); store at -20 °C

4. Trypsin-EDTA (0.25%), phenol red (Gibco, catalog number: 25200056); once thawed, store at 4 °C

5. PBS 10× (Eurobio, catalog number: ET330A)

6. Pyridostatin hydrochloride (PDS) (Sigma-Aldrich, catalog number: SML2690-5MG)

7. Dulbecco’s PBS 10× (DPBS) (Dutscher, catalog number: X0515-500)

8. TBS 10× (Euromedex, catalog number: ET220-B)

9. Triton X-100 (Sigma-Aldrich, catalog number: X100-100ML); store at 4 °C

10. Tween-20 (Fisher BioReagents, catalog number: BP337-500)

11. 1,4-Piperazinediethanesulfonic acid (PIPES) (Sigma-Aldrich, catalog number: P8203)

12. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 71376-1KG)

13. Sucrose (Sigma-Aldrich, catalog number: S0389)

14. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266)

15. RNase A 10 mg/mL (Thermo Scientific, catalog number: EN0531); store at -20 °C

16. TURBO^TM DNase kit 2 U/μL containing TURBO^TM DNase and 10× TURBO^TM DNase buffer (Invitrogen, catalog number: AM2238); store at -20 °C

17. Methanol (MeOH) (Carlo Erba Reagents, catalog number: 412383); store at -20 °C

18. Paraformaldehyde (PFA) 16% w/v aqueous solution, methanol free (Thermo Scientific, catalog number: 28908); once opened, store in a 15 mL Falcon tube at 4 °C

19. Bovine serum albumin (BSA) (Seqens, catalog number: 1000-70); store at 4 °C

20. Fish gelatin (Sigma-Aldrich, catalog number: G7041); store at 4 °C

21. MultiTASQ (Merck, catalog number: SCT247); store at -20 °C

22. ^azMultiTASQ (not commercially available, contact D. Monchaud); store at -20 °C

23. Alexa Fluor^TM 594 azide (AF-az) 1 mM in DMSO [Alexa Fluor^TM 594 carboxamido-(6-azidohexanyl), triethylammonium salt], mixed isomers (Invitrogen, catalog number: A10270); store at -20 °C, avoid light

24. Click-iT^TM Alexa Fluor^TM 594 sDIBO alkyne (DIBO-AF) 5 mM in DMSO (Invitrogen, catalog number: C10407); store at -20 °C, avoid light

25. Copper(II) sulfate (CuSO₄) (Sigma-Aldrich, catalog number: C7631-250G)

26. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896)

27. Sodium ascorbate (NaAsc) (Sigma-Aldrich, catalog number: 11140)

28. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, catalog number: D9542)

29. VECTASHIELD^® antifade mounting medium (VectorLab, catalog number: H1900); store at 4 °C

30. Transparent nail polish

Solutions

Note: Filtered solutions must be aliquoted to avoid cross-contamination and/or bacterial counterstaining with DNA intercalating dyes. Solutions with adjusted pH must be prepared with a sufficient volume of ultrapure water (ca. 90% of final volume) to resuspend powder by mixing, then pH must be adjusted, and finally, the solution can be completed with ultrapure water to final volume.

1. PDS 20 mM in DMSO

Note: Please refer to the manufacturer’s certificate of analysis to obtain the molecular weight of PDS with the number (y) of molecules of HCl as counterion and with the number (z) of molecules of water [final molecular weight (g/mol) = 596.64 + y × 36.46 + z × 18.02]. Store at -80 °C.

2. MultiTASQ or ^azMultiTASQ 10 mM in nuclease-free water

Note: Molecular weight of MultiTASQ: 1801.12 g/mol. Molecular weight of ^azMultiTASQ: 1846.16 g/mol. To avoid batch-to-batch variations in concentrations of stock solution of TASQ, we recommend measuring the absorbance of TASQ’s guanines at λ = 246 nm (see section A. Preparation of TASQ). Store at -20 °C.

3. AF-az 1 mM in DMSO

Note: Please refer to the manufacturer’s molecular weight depending on the number of triethylammonium molecules as counterion (molecular weight should be around 1,050 g/mol for 2 counterions). Store at -20 °C, avoid light.

4. DIBO-AF 5 mM in DMSO

Note: Please refer to the manufacturer’s molecular weight depending on the number of triethylammonium molecules as counterion (molecular weight should be around 1,200 g/mol for 2 counterions). Store at -20 °C, avoid light. Intermediate dilution of 0.5 mM of DIBO-AF in water (10% final concentration of DMSO) is more handful for SPAAC mix.

5. DAPI 1 mg/mL in water

Note: Store at 4 °C, avoid light.

6. Sterile PBS 1× (see Recipes)

7. Sterile supplemented DMEM (see Recipes)

8. Filtered DPBS 1× (see Recipes)

9. Filtered DPBS-Tx (see Recipes)

10. Filtered TBS-Tw (see Recipes)

11. PIPES 100 mM, pH 7.0 (see Recipes)

12. NaCl 1 M (see Recipes)

13. MgCl₂ 1 M (see Recipes)

14. Filtered sucrose 1 M (see Recipes)

15. Filtered cytoskeletal pre-extraction (CSK) buffer (see Recipes)

16. Filtered BSA blocking buffer (see Recipes)

17. Filtered FBS blocking buffer (see Recipes)

18. MultiTASQ 20 μM (200 μL) for post-fixation staining (see Recipes)

19. ^azMultiTASQ 20 μM (200 μL) for post-fixation staining (see Recipes)

20. CuSO₄ 100 mM (see Recipes)

21. IGEPAL CA-630 10% (see Recipes)

22. NaAsc 300 mg/mL (150×) (see Recipes)

23. CuAAC mix (see Recipes)

24. SPAAC mix (see Recipes)

Recipes

1. Sterile PBS 1× (500 mL)

Note: Prepare for cell culture (sterilize by filtration or autoclaving) and open only under sterile conditions.

2. Sterile supplemented DMEM (555 mL)

Note: Prepare directly in a DMEM bottle (from the manufacturer) and open only under sterile conditions.

3. Filtered DPBS 1× (500 mL)

Note: Filter and aliquot 50 mL for use. Keep a 50 mL aliquot at 4 °C as cold DPBS.

4. Filtered DPBS-Tx (500 mL)

Note: Filter and aliquot 50 mL for use.

*Tip needs to be cut due to Triton X-100 viscosity.

5. Filtered TBS-Tw (500 mL)

Note: Filter and aliquot 50 mL for use.

*Tip needs to be cut due to Tween-20 viscosity.

6. PIPES 100 mM, pH 7.0 (50 mL)

Note: Store at 4 °C up to a year. We recommend controlling pH if PIPES has not been used in the past 3 months.

*Add 40 mL of ultrapure water before adjusting pH. Once pH is adjusted, complete with water to obtain 50 mL of solution.

**pH equilibration with NaOH 10 M is necessary to dissolve PIPES; adjust pH to 7.0 by adding dropwise NaOH in solution under stirring.

7. NaCl 1 M (50 mL)

Note: Store at 4 °C for up to a year.

*Add 40 mL of ultrapure water and allow the solution to mix completely before adding any more water.

8. MgCl₂ 1 M (50 mL)

Note: Store at 4 °C for up to a year, avoid light.

*Add 40 mL of ultrapure water and allow the solution to mix completely before adding any more water.

9. Filtered sucrose 1 M (50 mL)

Note: Filter and store at -20 °C to avoid contamination.

*Add 30 mL of ultrapure water and allow the solution to mix completely before adding any more water as sucrose will take a substantial volume when fully dissolved.

10. Filtered cytoskeletal pre-extraction (CSK) buffer (50 mL)

Note: Filter with a 0.22 μm PES filter for a syringe and aliquot 5–10 mL of CSK in vials under sterile conditions. Store aliquoted vials at -20 °C. Avoid freeze/thaw cycles of aliquoted vials (2–3 cycles maximum).

*Tip needs to be cut due to Triton X-100 viscosity. Homogenize before adding Triton X-100 to avoid foaming.

11. Filtered BSA blocking buffer (500 mL)

Note: We found that the appropriate blocking buffer for MultiTASQ is BSA blocking buffer, and that for ^azMultiTASQ is FBS blocking buffer (see Troubleshooting 4; see Figure 10).

Filter and aliquot 50 mL of blocking buffer in vials under sterile conditions. Store aliquoted vials at -20 °C. Once a vial is thawed, store at 4 °C for 2–3 months maximum.

*Add 300 mL of water, allow BSA and fish gelatin to dissolve, and complete to 500 mL before addition of Triton X-100. The tip needs to be cut due to Triton X-100 viscosity. Homogenize before adding Triton X-100 to avoid foaming.

12. Filtered FBS blocking buffer (50 mL)

Note. We found that the appropriate blocking buffer for MultiTASQ is BSA blocking buffer, and that for ^azMultiTASQ is FBS blocking buffer (see Troubleshooting 4; see Figure 10).

Prepare and filter under sterile conditions with a 0.22 μm PES filter for a syringe. Store at 4 °C for 1–2 months maximum.

13. MultiTASQ 20 μM (200 μL) for post-fixation staining

Note: Prepare just before incubation with MultiTASQ (see Procedure for post-fixation staining). This volume corresponds to the volume required for a well (see General note 5). This solution cannot be stored.

14. ^azMultiTASQ 20 μM (200 μL) for post-fixation staining

Note: Prepare just before incubation with ^azMultiTASQ (see Procedure for post-fixation staining). This volume corresponds to the volume required for a well (see General note 5). This solution cannot be stored.

15. CuSO₄ 100 mM (1 mL)

Note: Store at 4 °C and avoid light.

16. IGEPAL CA-630 10% (1 mL)

Note: Store at 4 °C.

*Tip needs to be cut due to IGEPAL CA-630 viscosity.

17. NaAsc 300 mg/mL (150×) (300 μL)

Note: Aliquot vials of 30 μL and store at -20 °C. Avoid freeze/thaw cycle as NaAsc is sensitive to oxidation.

18. CuAAC mix (200 μL)

Note: Prepare just before “click” chemistry step with MultiTASQ (see Procedure). This volume corresponds to the volume required for a well (see General note 5). Add reagents in the order listed below. This solution cannot be stored.

NaAsc 30 mg/mL (or 15×) should be prepared in nuclease-free ultra-pure water before CuAAC mix; for 15 μL, add 1.5 μL of NaAsc 300 mg/mL to 13.5 μL of nuclease-free ultra-pure water.

19. SPAAC mix (200 μL)

Note: Prepare just before “click” chemistry step with ^azMultiTASQ (see Procedure). This volume corresponds to the volume required for a well (see General note 5). This solution cannot be stored.

Laboratory supplies

1. PES filter 0.22 μm Stericup system (Sigma-Aldrich, catalog number: S2GPU10RE)

2. Omnifix^TM solo 50 mL single-use syringe (Braun, catalog number: 4613503F)

3. PES filter 0.2 μm (Minisart^TM high flow) for syringe (Sartorius, catalog number: 16532----------K)

4. Tissue culture treated flask (75 cm²) (Falcon, catalog number: 353136)

5. Glass coverslips 13 mm, #1.5 (VWR, catalog number: 631-0150)

6. Microscope glass slides 26 × 76 × 1 mm (ISO8037/I) (Epredia, catalog number: AA00000112E01MNZ10)

7. 24-well polystyrene microplate (2 cm²) (Falcon, catalog number: 353047) or 4-well polystyrene microplate (2 cm²) (SPL Life Science, catalog number: 30004)

Equipment

1. pH meter

2. Spectrophotometer (UV5Nano)

3. Biological safety cabinet

4. 37 °C, 5% CO₂ incubator

Note: Equipment is dedicated to mammalian cell culture to avoid any bacterial contamination.

5. Aspiration system

Note: The aspiration system allows hazardous biological fluids (cell debris, fixative reagent, DAPI, etc.) to be disposed of.

6. Counting chamber (Malassez pattern)

7. Phase-contrast or brightfield microscope

8. Widefield microscope (Olympus, model: IX83)

a. DAPI filter set (Chroma, catalog number 49000)

b. TexasRed filter set (Chroma, catalog number 49306)

c. Optional for multiplexing: GFP filter set (Chroma, catalog number 49002) and/or Cy5 filter set (Chroma, catalog number 49009) and/or Cy7 filter set (Chroma, catalog number 49007)

Software and datasets

1. CellSens Dimension (Olympus, v3.2)

2. FIJI (ImageJ, v2.14.0)

3. Excel (Microsoft, v16.77.1)

4. Prism (GraphPad, v10.3.1)

5. Code has been deposited to GitHub (see Data analysis)

Procedure