Materials and Reagents

Note: The materials below can also be substituted for equivalent reagents from other companies. Cell culture media and additives will vary based on the cells to be used for the assay. For our protocol, we have used primary dermal fibroblasts from neonatal mice and the mast cell line MCP-5.

1. Cell culture and counting
   
   1. 70% ethanol
   2. Recombinant mouse IL-3 (Thermo Fisher Scientific, Gibco^TM, catalog number: PMC0034 )
      Note: Lyophilized mIL-3 should be stored at 4 °C. Aliquot and store reconstituted mIL-3 at ≤ -20 °C, use within 12 months of reconstitution.
   3. Dulbecco’s Modified Eagle’s Medium (DMEM) (HiMedia Laboratories, catalog number: AL066A )
      Note: Store at 4 °C for up to 12 months.
   4. Fetal Bovine Serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 26140095 )
      Note: Store at -20 °C for up to 12 months.
   5. Sodium Pyruvate (Thermo Fisher Scientific, Gibco^TM, catalog number: 11360070 )
   6. Non-Essential Amino Acids (Thermo Fisher Scientific, Gibco^TM, catalog number: 11140050 )
   7. RPMI media (Sigma-Aldrich, catalog number: R0883 )
      Note: Store at 4 °C. Media is light sensitive, use within 12 months of manufacturing.
   8. Solution of penicillin-streptomycin (10,000 U/ml penicillin G sodium and 10,000 μg/ml streptomycin sulfate, Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
      Note: Use within 12 months of manufacturing.
   9. Solution of trypsin, 2.5% 10x stock (HiMedia Laboratories, catalog number: TCL051 )
      Note: Use within 12 months of manufacturing.
   10. Trypan Blue stain (Thermo Fisher Scientific, Gibco^TM, catalog number: 15250061 )
       Note: Use within 3 years of manufacturing.
   11. 1x PBS (see Recipes) (Cold Spring Harbor, doi:10.1101/pdb.rec556)
       


2. Collagen lattice
   
   1. 10 μl, 200 μl and 1,000 μl pipette tips (any desired make)
   2. 0.22-μm syringe-driven filter units (Merck, catalog number: SLGV033RS )
      
   3. 10 ml Syringes (Dispo Van)
   4. 24-well cell culture plates (Eppendorf, catalog number: 0030722116 )
   5. 15 ml Graduated Centrifuge Tubes, sterilized (Tarsons, catalog number: 546021 )
   6. 1.5 ml microfuge tubes (Corning, Axygen^®, catalog number: MCT-150-C )
      Note: Need to be autoclaved/sterilized before use.
   7. Rat tail collagen type 1 (Merck, catalog number: 08-115 )
      Note: Store at 4 °C, use within 12 months of manufacturing.
   8. Glacial Acetic acid 99.8% (Sigma-Aldrich, catalog number: 109088 )
      Note: Store at 15-30 °C.
   9. NaOH pellets (Sigma-Aldrich, catalog number: S8045 )
      Note: Store at 15-30 °C.
      
   10. Stock collagen solution (3 mg/ml) (see Recipes)
   11. 1 M NaOH Solution (see Recipes)
   12. Acetic acid solution (0.1%) (see Recipes)
   
   
3. Image acquisition/analysis
   
   1. Parafilm (Bemis, catalog number: PM996 )
   2. Crystal Violet stain (Optional; Sigma-Aldrich, catalog number: C3886 )
      Note: Store at 15-30 °C.

Equipment

1. 10 μl, 200 μl and 1 ml pipettes (any desired make)
2. Centrifuge (Eppendorf, model: 5810 R or equivalent)
3. Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
4. Incubator (CO₂ incubator, Thermo Fisher Scientific, model: Forma^TM Steri-Cycle^TM , or equivalent)
5. Microscope (OLYMPUS, model: SZX16 or equivalent)
6. Digital Camera 3.2 megapixel (Nikon, model: COOLPIX A10 or equivalent) and stand with camera holder
7. Flatbed scanner (Epson, model: V39 or equivalent)
   

Software

1. ImageJ Software v. 1.8.0 (National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/download.html) (Schindelin et al., 2012; Rueden et al., 2017)

Procedure