Materials and reagents

Biological materials

1. DMS114 human small cell lung cancer (SCLC) cells [American Type Culture Collection (ATCC), catalog number: CRL-2066]. The cells should be grown to 70%–80% confluence in DMS114 culture media (see Recipe 3) and kept in a humidified environment at 37 °C (in the presence of 5% CO₂) in a cell culture incubator [10]. Although we are describing the BCA protocol using DMS114 human SCLC cells, the pro-invasive activity of any relevant cancer cell line may be measured using this assay. Normal cells display pro-invasive activity under specific biological conditions. Such conditions may include treatment with growth factors, steroids, and carcinogenic chemicals. The BCA may be used to measure the effect of such chemicals on the invasion of normal cells.

1. Corning^TM BioCoat^TM Matrigel^TM invasion chamber inserts coated with Matrigel matrix. These invasion chambers are sold in two formats. The first is a 6-well format (Fisher Scientific, catalog number: 08-774-121). The second is a 24-well format (Fisher Scientific, catalog number: 08-774-122). Store at -20 °C. We use the 24-well format for the protocol described in the present manuscript

2. Sterile bovine serum albumin (BSA) solution at a concentration of 30% (w/v) in DPBS (Millipore-Sigma, catalog number: A7596)

3. Roswell Park Memorial Institute (RPMI)-1640 medium (ATCC, catalog number: 30-2001). Store at 4 °C, 1 M HEPES (Thermo Fisher, catalog number: 15630080)

4. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020). Long-term storage for FBS is at -70 °C. In our laboratory, FBS is aliquoted into 50 mL centrifuge tubes and stored at -20 °C. The FBS aliquot is thawed and stored at 4 °C for immediate use

5. Trypsin-EDTA (0.25% Trypsin, 0.53 mM EDTA) (ATCC, catalog number: 30-2101). Long-term storage for Trypsin-EDTA is at -20 °C. However, Trypsin-EDTA is aliquoted and stored at 4 °C for immediate use

6. 1 M HEPES (Thermo Fisher Scientific, catalog number 15630080). Long-term storage for 1 M HEPES is at -20 °C. However, HEPES is aliquoted and stored at 4 °C for immediate use

7. Penicillin-streptomycin solution (ATCC, catalog number: 30-2300)

8. Dulbecco’s phosphate buffered saline (PBS) without calcium and magnesium (Corning, Fisher, catalog number: 21-031-CM). Store at 4 °C

9. Corning cell counting chamber (Fisher Scientific, catalog number: 07-200-988)

10. Capsaicin (Sigma-Aldrich, catalog number: M2028-50MG). Capsaicin is the hot and spicy ingredient of chili peppers. It is a potent agonist of transient receptor potential cation channel subfamily V member 1 (TRPV1). Store at 4 °C

11. PP2 is a potent and selective inhibitor of the Src family tyrosine kinases [11]. PP2 was manufactured by Enzo Biosciences (Enzo Biosciences, catalog number: 50-201-0680) and obtained from Thermo Fisher Scientific. The PP2 is delivered as powder and stored at -20 °C. The PP2 is dissolved in DMSO (Corning DMSO, Fisher, catalog number: MT-25950CQC) at a final concentration of 10 mM. The PP2 solution is aliquoted into microfuge tubes and stored at -20 °C

12. Dimethyl sulfoxide (DMSO), ACS certified (Fisher BioReagents, catalog number: D128-500). Store at room temperature

13. Aqua Solutions crystal violet 1% w/v aqueous (Fisher Scientific, catalog number: NC9002731). Store at room temperature

1. Stock capsaicin (100 mM) (see Recipes)

2. WS-CAP-100X (2 mM) (see Recipes)

3. VEH-CAP-100X (see Recipes)

4. Stock PP2 (10 mM) (see Recipes)

5. WS-PP2-100X (500 µM) (see Recipes)

6. VEH-PP2 (see Recipes)

7. Crystal violet staining solution (see Recipes)

8. DMS114 culture medium (see Recipes)

9. RPMI-BSA (see Recipes)

Recipes

1. Stock capsaicin (100 mM), 1 mL

   Weigh 30.5 mg of capsaicin in a sterile autoclaved amber-colored 1.5 mL microfuge tube. In a laminar flow hood, add 1 mL of DMSO to the tube containing capsaicin. Vortex to mix. Aliquot in sterile amber-colored 1.5 mL microfuge tubes. We typically put 25 µL of capsaicin stock solution in each aliquot. Store the aliquots at -20 °C. The aliquots are stable for one month at -20 °C. Laboratory personnel should wear gloves, mask, and face goggles when working with powdered capsaicin. Capsaicin can cause intense irritation if it comes in contact with the skin, face, or eyes.




2. WS-CAP-100X (2 mM), 100 µL

   Take out an aliquot of 100 mM capsaicin solution from -20 °C. Put the tube in a 37 °C water bath for 5 min (until the entire solution is thawed). Vortex briefly to mix. Add 2 µL of 100 mM stock capsaicin to 98 µL of RPMI-BSA (in an autoclaved amber-colored 1.5 mL microfuge tube) in a laminar flow hood. Vortex to mix. Keep the solution on ice until used in the experiment. We make a fresh solution of WS-CAP before every assay and discard any leftover solution.




3. VEH-CAP-100X, 100 µL

   Add 2 µL of DMSO to 98 µL of RPMI-BSA (in an autoclaved amber-colored 1.5 mL microfuge tube) in a laminar flow hood. Vortex to mix. We do not store the VEH-CAP-100X solution. We make a fresh solution of VEH-CAP before every assay and discard any leftover solution.




4. Stock PP2 (10 mM), 333 µL

   Add 333 µL of DMSO to the bottle of 1 mg of PP2 in a laminar flow hood. Vortex to mix. Aliquot in autoclaved amber-colored 1.5 mL microfuge tubes (we put 25 µL of PP2 solution in each aliquot). Store the aliquots in -20 °C. The aliquots are stable for one month at -20 °C.




5. WS-PP2-100X (500 µM), 1 mL

   Take out an aliquot of 10 mM PP2 solution from -20 °C. Put the tube in a 37 °C water bath for 5 min (until the entire solution is thawed). Vortex briefly to mix. Add 5 µL of stock solution of DMSO to 95 µL of RPMI-BSA (in an autoclaved amber-colored 1.5 mL microfuge tube) in a laminar flow hood. Vortex to mix. Keep the solution on ice until it is used for the experiment. We make a fresh solution of WS-PP2 before every assay and throw away the leftover solution.




6. VEH-PP2, 1 mL

   Add 5 µL of DMSO to 95 µL of RPMI-BSA (in an autoclaved amber-colored 1.5 mL microfuge tube) laminar flow hood. Vortex to mix. We make a fresh solution of vehicle before every assay and do not store it.




7. Crystal violet staining solution, 20 mL

   Add 5 mL of 1% crystal violet solution to 15 mL of Milli Q water. Vortex to mix. Store the solution at room temperature.




8. DMS114 culture medium

   DMS114 cells were cultured following the published protocols [10]. All procedures are performed in a laminar flow hood. The DMS114 culture medium consists of RPMI-1640 with 1% Penicillin streptomycin solution (containing 100 units/mL penicillin, 50 µg/mL streptomycin), 25 mM HEPES, and 10% FBS. ATCC RPMI-1640 (30-2001 formulation) comes formulated with 10 mM HEPES. We then add an additional 7.5 mL of 1 M HEPES to 500 mL of RPMI-1640 to obtain a 25 mM concentration of HEPES. Store the media at 4 °C. To note, many laboratories add 50 mL of FBS to the full bottle of media to obtain 10% (v/v). We do not typically add FBS to the entire RPMI bottle. The rationale for this is to facilitate the most efficient use of each RPMI bottle. Many of our experiments require serum-free media, so we add the FBS as needed. The cells should be grown to 70%–80% confluence using the media described above and kept in a sterile cell culture incubator with a humidified environment, set at 37 °C with 5% CO₂.

   DMS114 culture medium	Final concentration in media	Quantity or Volume added to 500 mL media
   RPMI-1640	NA	500 mL
   Penicillin-streptomycin solution	100 units/mL penicillin, 50 µg/mL streptomycin	5 mL
   HEPES	25 mM	7.5 mL
   FBS	10% v/v	50 mL




9. RPMI-BSA

   Add 100 µL of BSA from the bottle containing 30% BSA solution to 30 mL of serum-free RPMI-1640 (at room temperature) in a sterile 50 mL tube. Mix gently to avoid air bubbles. All these procedures should be performed in the laminar flow hood. We make fresh RPMI-BSA before each experiment. Store at room temperature for the duration of the assay.

   DMS114 culture medium	Final concentration in media	Quantity or Volume
   RPMI-1640	NA	30 mL
   30% BSA solution	0.1% v/v	50 µL

Laboratory supplies

1. Nunc^TM EasYFlask^TM T-75 cell culture flasks (Thermo Scientific, catalog number: 156499)

2. Sterile Corning 24-well polystyrene tissue culture insert companion plate with lid (Corning Life Sciences, catalog number: 353504)

3. 96-well plate, non-treated surface, pack of 25 (Thermo Scientific, catalog number: 12-566-202)

4. Puritan^TM sterile DNA-free standard cotton swab, wood handle (Fisherbrand, catalog number: 22-025-201)

5. Sterile 15 mL polypropylene centrifuge tubes (Thermo Scientific, Nunc, catalog number: 12-556-017)

6. 1.5 mL natural microcentrifuge tubes (Fisherbrand Premium, catalog number: 05-408-129). Autoclave before use

7. 1.5 mL amber-colored microcentrifuge tubes (Fisherbrand Premium, catalog number: 05-408-134). Autoclave before use

8. Nunc 15 mL conical sterile polypropylene centrifuge tubes with plastic racks (Thermo Scientific, catalog number: 12-565-269)

9. Nunc 50 mL conical sterile polypropylene centrifuge tubes with plastic racks (Thermo Scientific, catalog number: 12-565-271)

10. Low-retention 2 mL microcentrifuge tubes (Fisherbrand, catalog number: 02-681-332)

11. Curved medium point stainless-steel general-purpose forceps (Fisherbrand, catalog number: 16-100-110). Autoclave the forceps before use with the Boyden chamber assay

12. Fine point high-precision forceps (Fisherbrand, catalog number: 22-327379). Autoclave the forceps before use with the Boyden chamber assay

Equipment

1. NU-540 laminar-flow biosafety cabinet (NuAire, Plymouth, MN, model: LabGard^® ES NU-540 Class II, Type A2)

2. Cell culture incubator maintained at 37 °C and 5% CO₂ (Thermo Scientific, Waltham, MA, model: Heracell VIOS 150i)

3. Microplate reader (Agilent, model: BioTek Gen 5)

4. Benchtop centrifuge (ThermoElectron Corporation, model: IEC Centra CL2)

5. Gilson PIPETMAN Pipettes [Marshall Scientific, catalog number: F144059M (P1000), F144058M (P200), F144056M (P10), F144054M (P2)]

Software and datasets

1. Agilent BioTek Gen 5 microplate reader and imager software

2. GraphPad Prism version 10.2.1

Procedure