Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin

Materials and reagents

Biological materials

1. Mammalian cells in culture: HeLa (ATCC^® CCL-2^TM)

Reagents

1. DMEM, high glucose, no glutamine, no phenol red (Gibco, catalog number: 31053044), stored at 4 °C

2. Fetal bovine serum (FBS) (Sigma, catalog number: F7524), stored at -20 °C until use

3. GlutaMAX I, 200 mM (Gibco, catalog number: 35050038), stored at 4 °C

4. Gentamicin (50 mg/mL) (Gibco, catalog number: 15750037), stored at -20 °C until use

5. Dulbecco's phosphate-buffered saline (DPBS), without calcium and magnesium (Gibco, catalog number: 14190250), stored at 4 °C

6. Trypsin-EDTA 0.5%, no phenol red (Gibco, catalog number: 15400054), stored at 4 °C

7. Trypan blue stain 0.4% (Gibco, catalog number: 15250061), stored at 4 °C

8. Pierce 16% formaldehyde (w/v), methanol-free (Thermo Scientific, catalog number: 28908), stored at room temperature (RT)

9. Tris(hydroxymethyl)aminomethane (Tris) (Sigma, catalog number: 252859), stored at RT

10. Sodium acrylate 97% (Sigma-Aldrich, catalog number: 408220), stored at RT inside a desiccator

11. Acryloyl-X SE, >95.0% (AcX) (TCI America, catalog number: A345025MG), stored at -20 °C

12. Guanidine hydrochloride 99.5% (Thermo Scientific, catalog number: 364791000), stored at RT

13. Poly-L-lysine solution 0.1% (w/v) in H₂O (Sigma-Aldrich, catalog number: P8920), stored at RT

14. Triton X-100 98%, DNAse free, RNAse free, protease free (Thermo Scientific, catalog number: 327371000) stored at 4 °C

15. Sigmacote (Sigma-Aldrich, catalog number: SLCM2185), siliconizing reagent for providing hydrophobic properties and reducing nonspecific adsorption; stored at 4 °C

16. 4-Hydroxy-TEMPO 97% (Sigma-Aldrich, catalog number: 176141), stored at RT

17. Proteinase K (New England Biolabs, catalog number: P81075), stored at -20 °C

18. Ethylenediaminetetraacetic acid (EDTA), 0.5 M) (Thermo Scientific, catalog number: J15694-AE) stored at RT

19. DAPI (4',6-diamidino-2-phenylindole, dihydrochloride, 10 μM in DMSO) (Invitrogen, catalog number: D1306) stored at -20 °C

20. Fluorescent phalloidin ExM reagent (TRITON); synthesized in-house according to [8] or commercially available as Actin ExM 532 (Chrometra Scientific, catalog number: ER-10-4-S), stored at -20 °C until use

21. Phosphate-buffered saline (PBS) 1× pH 7.4, without calcium, magnesium, and phenol red (Gibco, catalog number: 10010023)

22. Phosphate-buffered saline (PBS) 10× pH 7.4, without calcium, magnesium, and phenol red (Gibco, catalog number: P7059)

23. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678)

24. Ammonium chloride (NH₄Cl) (Sigma-Aldrich, catalog number: 213330)

25. Hydrochloric acid 37% (HCl) (Sigma-Aldrich, catalog number: 258148)

26. N,N,N',N'-Tetramethyl ethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T7024)

27. N,N'-Methylenebisacrylamide (Sigma-Aldrich, catalog number: 146072)

28. Acrylamide (Sigma-Aldrich, catalog number: A8887)

29. Sodium chloride (Sigma-Aldrich, catalog number: S9888)

30. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650)

Solutions

1. Gelation stock (polymerization, 10 mL) (see Recipes)

2. 4-hydroxy-TEMPO stock solution (see Recipes)

3. Catalyst (see Recipes)

4. Active monomer solution (200 μL) (see Recipes)

5. Digestion solution (see Recipes)

6. AcX (acryloyl-X) (see Recipes)

7. 1 M HCl solution (see Recipes)

8. 100 mM NH₄Cl solution (see Recipes)

9. 5 M NaCl solution (100 mL) (see Recipes)

10. 4 M Guanidine HCl (100 mL) (see Recipes)

11. 1 M Tris, pH 8 (100 mL) (see Recipes)

Recipes

General note: Some reagents, such as those involved in polymerization or gelation steps, should be prepared just before use to ensure optimal activity and avoid degradation.

1. Gelation stock (polymerization, 10 mL)

Note: Prepare aliquots of 188 μL (polymerization for one sample) and store at -20 °C.

2. 4-hydroxy-TEMPO stock solution

0.5 g per 100 mL of MQ water.

Prepare aliquots and store at -20 °C.

3. Catalyst

Prepare aliquots and store at -20 °C.

Prepare aliquots and store at -20 °C.

4. Active monomer solution (200 μL)

Note: Keep the solution on ice until ready to use. Add APS and TEMED (see Recipe 3) just before use.

5. Digestion solution

Note: The 1 M stock solution of Tris was adjusted to pH 8 using 37% HCl.

6. AcX (acryloyl-X)

a. To prepare AcX stock solutions, dissolve AcX in anhydrous DMSO to a concentration of 10 mg/mL and store 10 μL aliquots at -20 °C.

b. Thaw the 10 mg/mL AcX stock to RT and dilute it 1:100 to a final concentration of 0.1 mg/mL by adding 990 μL of 1× PBS.

7. 1 M HCl solution

To prepare the desired concentration of HCl from the stock solution (typically 37%), dilute it by mixing 8.3 mL of concentrated HCl with 91.7 mL of MQ water.

8. 100 mM NH₄Cl solution

a. Measure 0.534 g of NH₄Cl.

b. Dissolve it in approximately 90 mL of PBS.

c. Adjust the final volume to 100 mL with PBS.

d. Store at 4 °C for short-term use or aliquot and freeze for long-term storage.

9. 5 M NaCl solution (100 mL)

a. Weigh 29.2 g of NaCl using an analytical balance.

b. Add the NaCl to approximately 80–90 mL of MQ water in a volumetric flask.

c. Once the NaCl has fully dissolved, add MQ water to bring the final volume to 100 mL.

d. Stir the solution thoroughly to ensure uniformity.

e. Store the 5 M NaCl solution at room temperature

10. 4 M guanidine HCl (100 mL)

a. Measure 38.212 g of guanidine HCl using an analytical balance.

b. Dissolve it in approximately 90 mL of MQ water in a volumetric flask.

c. Adjust the final volume by adding MQ water to 100 mL.

d. Mix the solution thoroughly and store the 4 M guanidine HCl solution a 4 °C.

11. 1 M Tris (pH 8) (100 mL)

a. Weigh out 12.1 g of Tris base using an analytical balance.

b. Add 80–90 mL of MQ water to dissolve it and stir the solution until the Tris base is completely dissolved.

c. Slowly add 1 M HCl (Recipe 7) to adjust the pH to exactly 8.0. Add the HCl drop by drop, stirring continuously to avoid overshooting the pH.

d. After adjusting the pH, transfer the solution to a 100 mL volumetric flask.

e. Swirl the solution gently to ensure it is homogeneous and store the 1 M Tris solution at 4 °C.

Laboratory supplies

1. T75 flask for cell culture (Thermo Fisher Scientific, catalog number: 156800)

2. 22 × 22 mm, #1.5H coverslips (https://www.carlroth.com/be/en/coverslips/coverslips-thickness-1-5/p/kcy1.1)

3. Microscope slides 26 × 76 mm, single concave (see Figure S2) (Pearl, catalog number: 7103)

4. Microscope slides 26 × 76 mm (https://www.galvoptics.co.uk/optical-components/microscope-slides/microscope-slides/?utm_source=google&utm_medium=cpc&utm_campaign=Galvoptics&utm_content)

5. 6-well plates (Thermo Fisher Scientific, catalog number: 150239)

6. Dumont HP crossover tweezers N7, Dumoxel 0.17 × 0.10 mm tip (Dumont, catalog number: AGT5007)

7. Microcentrifuge tubes (Eppendorf, catalog numbers: 0030121023, 0030120086)

8. Conical tube 15 mL/50 mL (Greiner Bio-One, catalog number: 188271/227261)

9. Cytosmart counting chamber with cover glass (Cytosmart, Corning, catalog number: 0699910591)

10. P1000 regular pipette tips (Thermo Scientific Finnpipette, catalog number: 94060717)

11. P200 regular pipette tips (Thermo Scientific Finnpipette, catalog number: 94060317)

12. P10 regular pipette tips (Thermo Scientific Finnpipette, catalog number: 94060117)

13. Spatula (Bochem, catalog number: 3101)

14. Parafilm (Merck, catalog number: P7793)

15. Scissors

16. 500 mL beaker

17. Photo glue (Collall, catalog number: COLFO0100)

18. 6-well glass bottom plate (confocal imaging dish) (see Figure S6) (Cellvis, catalog number: P06-1.5H-N)

Equipment

1. 37 °C incubator with 5% CO₂ in a humidified environment (Thermo Scientific, model: HERACELL VIOS 160i)

2. Cytosmart cell counter (Corning, catalog number: 6749)

3. Incubator INCU-Line^® IL 10 (VWR, catalog number: 390-0384)

4. Microscope (Leica Microsystems GmbH, model: TCS SP8)

5. Objective lenses HC PL APO CS2 63×/1.20 WATER and HC PL APO CS2 20×/0.75 DRY

Software and datasets

1. Fiji (ImageJ, https://imagej.net/software/fiji/) [24]

2. LAS X (https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/)

3. BioRender (https://www.biorender.com/) was used for the Graphical overview: https://app.biorender.com/illustrations/66d5e6f134b972cb4b0abed5?slideId=505c1415-eac1-400b-83fe-dd8171f5f3d3

Procedure