Gelcount system

5.0 × 10³ cells were seeded in each well of six-well plates. 14 days later, cells were subsequently washed with PBS and fixed with cold methanol, stained with 0.05% crystal violet (Sigma-Aldrich), photographed and counted using the GelCount System (Oxford Optronix) and the GelCount^TM operating software.

Cellular COX activity was measured by stimulation with 50 μM ¹⁴C-arachidonic acid (30 s at 37°C) followed by quantification of radiolabeled prostaglandin products, as described [62 (link)]. The percentage of total products was expressed per 10⁶ cells. Cellular secretion of VEGF into serum-free medium with and without stimulation with arachidonic acid (20 μM) for 24 hours was measured using a human VEGF ELISA plate according to manufacturer's instructions (Signosis Inc, Santa Clara, CA). Sulfurhodamine B (SRB) growth assays were performed as previously described [31 (link), 69 (link)]. Effects on cell growth were measured 72 hours after plating of cells. Clonogenic assays were performed and quantified using the GelCount System (Oxford Optronix, UK) in the DHSR at VUMC [69 (link)]. Cell invasion assays were performed using low basement membrane cell invasion inserts according to manufacturer's instructions (Trevigen, Gaithersburg, MD). Standard 10% FBS growth medium was used as a chemoattractant in the lower chamber for cells grown on the inserts. Following invasion, cells on the side of the insert facing the lower chamber were stained with 1% crystal violet, and the insert carefully mounted on a microscope slide for quantification of invading cells. 5 fields per insert were counted using a 5x objective lens.

As described previously^{6 (link)}, mammosphere formation assay was performed as previously described^{6 (link)}. Single-cell suspensions of SUM149 cells (2 × 10⁴ cells/well) and BT549 cells (1 × 10⁴ cells/well), in MammoCult Human Medium Kit (StemCell Technologies, Vancouver, Canada), were seeded in 6-well ultra-low attachment plates (Corning Incorporated Costar, Corning, NY, USA). After a 7-day incubation, mammospheres were stained using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma), and spheres greater than 80 μm in diameter were counted using the GelCount system (Oxford Optronix, UK) according to the manufacturer’s instructions.

As described previously^{6 (link)}, a bottom agarose layer (1%) was laid in 12-or 6-well plates. Cells (1 × 10⁴ cells/well), resuspended in 2 mL of 0.5% agarose solution in complete medium, were overlaid and incubated for 25 days. Colonies formed were stained using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma), and those greater than 80 μm in diameter were counted using the GelCount system (Oxford Optronix, UK) according to the manufacturer’s instructions.

Sulforhodamine B (SRB) colorimetric assay was performed to evaluate clonogenicity after treatment with our ADCs according to a published protocol^{53 (link)}. HCC1954 and HCC1954-TDR cells were plated into 24-well plates (1000 cells per well) and incubated overnight. The cells were treated with each ADC for 5 days. Subsequently, cells were fixed with 5% trichloroacetic acid and then stained with 0.03% of SRB solution (Sigma) at room temperature for 30 min. The stained cells were imaged using a GelCount system (Oxford Optronix) and then dissolved in Tris buffer (10 mM). Optical density was determined fluorometrically using a VICTOR X3 plate reader (Ex: 488 nm, Em: 585 nm).

The soft agar assay is a good predictor of in vivo activity in that a positive result is a potential indicator of in vivo carcinogenesis. It is also the most stringent assay for detecting malignant transformation of cells. For each of the listed TNBC and HER2+ breast cancer cell lines, cells (2 to 5 × 10³ cells/well) in 2 mL of 0.4% agarose solution were overlaid onto the bottom agar layer (0.7%) in 12-well plates. The plates were incubated for 3 to 6 weeks with or without the drug, and colonies were stained with 200 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (2 mg/mL; #AAL1193906, Fisher Scientific) for 3 h at 37 °C in 5% CO₂. According to the manufacturer’s instructions, stable colonies greater than 80 μm in diameter were counted using a GelCount system (Oxford Optronix, Milton, UK).