Actin Quantification Using the Filamentous Actin Segmentation Tool (FAST)

Materials and reagents

Biological materials

1. HeLa cells (ATCC CCL-2^TM)

Reagents

1. Myosin X antibody with mouse host (Novus Biologicals, catalog number: NBP2-88926), store at -20 °C

2. Human myosin IIA (GeneTex, catalog number: GTX33939), store at -20 °C

3. Anti-paxillin antibody (Y113) with rabbit host (Abcam, catalog number: ab32084), store at -20 °C

4. Phalloidin-iFluor 647 reagent (Abcam, catalog number: ab176759), store at -20 °C

5. Goat anti-mouse IgG (H+L) (Alexa Fluor^® 405) (Life Technologies Inc, catalog number: A31553), store at 4 °C

6. Goat anti-human IgG (H+L) (Alexa Fluor^® 488) (Life Technologies Inc, catalog number: A11013), store at 4 °C

7. Goat anti-rabbit IgG H&L (Alexa Fluor^® 568) (Abcam, catalog number: ab175471), store at -20 °C

8. Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L D-Glucose and phenol red (Gibco, catalog number: 11965118), store at 4 °C

9. Phosphate-buffered saline (PBS), 1× with calcium and magnesium (Corning, catalog number: 21-030-CV), store at 4 °C

10. Trypsin-EDTA solution, 0.25% (Gibco, catalog number: 25200056), store at 4 °C

11. Fetal bovine serum (FBS), heat-inactivated (Thermo Scientific^TM, catalog number: 10270106), store at -20 °C

12. Penicillin-streptomycin 100× solution (Cytiva, catalog number: SV30010), store at -20 °C

13. PIPES, sodium salt (1.5) reagent grade (BioShop, catalog number: PIP663), store at room temperature

14. Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), ultra-pure, min. 98% (BioShop, catalog number: EGT101), store at room temperature

15. Magnesium chloride (MgCl₂) (Quality Biological, catalog number: 351-033-721EA), store at room temperature

16. Triton^TM X-100, pH 9.7, non-ionic, liquid (Sigma-Aldrich, catalog number: T8787-50ML), store at room temperature

17. Fibronectin (Corning, catalog number: 354008), store at 4 °C

18. Sucrose (Ward’s Science, catalog number: 470302–808), store at room temperature

19. Pierce^TM 16% formaldehyde (PFA) (w/v), methanol-free (Thermo Scientific^TM, catalog number: 28908), store at room temperature

20. HyClone^TM HyPure water, cell culture grade (Cytiva, catalog number: SH30529.03), store at room temperature

21. Bovine serum albumin (BSA), BioReagent (Sigma-Aldrich, catalog number: A9418), store at -20 °C

22. Sodium azide solution (Sigma-Aldrich, catalog number: 08591-1ML-F), store at 4 °C

Solutions

1. Cell culture medium (see Recipes)

2. Cytoskeletal buffer (see Recipes)

3. Fixing agent (see Recipes)

4. Permeabilization solution (see Recipes)

5. Blocking solution (see Recipes)

6. Sodium azide solution (see Recipes)

7. Primary antibody solution (see Recipes)

8. Secondary antibody solution (see Recipes)

Recipes

1. Cell culture medium

Supplement 450 mL of DMEM with 50 mL of heat-inactivated FBS and 5 mL of penicillin-streptomycin. Mix gently to avoid foaming. Store at 2–8 °C for 2–4 weeks. Use sterile technique.

2. Cytoskeletal buffer

Mix 30–35 mL of dH₂O with 1.21 g of PIPES, 95 mg of EGTA, and 20.3 mg of MgCl₂. Raise the pH to 6.8 using KOH. Make the final volume of solution to 50 mL by adding dH₂O. Sterilize using a 0.22 μm filter. Store at 2–8 °C.

3. Fixing agent

Add 1 g of sucrose to 1 mL of cytoskeletal buffer and mix well with 6.5 mL of culture-grade water. Finally, add 2.5 mL of 16% PFA aliquot and mix gently to avoid foaming. Store at 2–8 °C for 2–4 weeks.

4. Permeabilization solution

Make permeabilization solution by adding 10 mL of PBS to 10 μL of Triton^TM X-100. Mix gently to avoid foaming. Store at 2–8 °C for 2–4 weeks.

5. Blocking solution

Make blocking solution by adding 100 μL of 200 mg/mL BSA stock to 10 mL of PBS and mix gently to avoid foaming. Store at 2–8 °C for 2–4 weeks.

6. Sodium azide solution

Supplement 1 mL of PBS with 1 μL of sodium azide. Store at 2–8 °C for 2–4 weeks. Use sterile technique.

7. Primary antibody solution

Make primary antibody solution by adding 1 μL of each of the antibodies listed above to 200 μL of blocking solution and mix gently to avoid foaming.

8. Secondary antibody solution

Make secondary antibody solution by adding 1 μL of each of the antibodies listed above to 200 μL of blocking solution and mix gently to avoid foaming.

Laboratory supplies

1. Cell culture flask with filter cap, 25 cm² (FroggaBio, catalog number: FB-T25-200)

2. 0.22 um PES syringe filter, 25 mm, sterile (FroggaBio, catalog number: SF0.22PES)

3. 8-well chambered cover glass with #1.5 high performance cover glass, 57 mm × 25 mm base (Cellvis, catalog number: C8-1.5H-N)

4. FroggaBio 15 mL conical tubes (rack) (FroggaBio, catalog number: TR15-500)

Equipment

1. Microscope (Nikon Eclipse Ti2 inverted microscope utilizing a CrestOptics X-Light V3 spinning disk integrated with a Photometrics Kinetix camera with 60× 1.2 Numerical Aperture Plan Apo VC water immersion objective)

2. Class II biological safety cabinet (ESCO)

3. CO₂ incubator (Thermo Scientific, model: 3110)

4. Freezer, -20 °C

5. Refrigerator, 2–8 °C

Software and datasets

Dataset, model, and software are provided in Table 1 along with version and license information. Additionally, Google Colab notebooks are provided for training to simulate a GUI on a cloud computing platform. Inference and preprocessing are performed with Ilastik [13] and Fiji/ImageJ [14], respectively.

Note: While FAST can be run freely on Google Colab, the availability of GPU resources on the cloud is subject to usage and the subscription type of the user.

Table 1. Software and datasets for data analysis. FAST can be downloaded from GitHub (https://github.com/Carleton-CTE-Lab/FAST-Protocol/archive/refs/heads/main.zip), and the corresponding model link is available on the Google Colab notebook. Dataset is publicly available on Zenodo (https://zenodo.org/records/18135376).

Procedure