Cyquant gr dye

For investigation of anchorage-independent cell growth soft-agar assays were performed using the CytoSelect 96-Well Cell Transformation Assay Kit (Cell Biolabs, Inc.) according to the manufacturer's instructions. In brief, 2500 cells per well were seeded in soft-agar in a 96-well plate and incubated at 37°C in 5% CO₂ with the indicated inhibitors. For siRNA experiments, cells were transfected with the indicated siRNAs 2 days prior to seeding equal cell numbers into the 96-well format in soft-agar, followed by drug treatment. After 7 days, agar was solubilized and cells were lysed according to the manufacturer's instructions. Colony formation was quantified using the fluorescent cell stain CyQUANT GR Dye (Cell Biolabs Inc.) in the Synergy HT Plate reader using Gen5 software from BioTek Instruments Inc.

For investigation of anchorage-independent cell growth, soft-agar assays were performed using the CytoSelect 96-Well Cell Transformation Assay Kit (Cell Biolabs, Inc.) according to the manufacturer's instructions. In brief, LN229 or 83Mes cells were seeded in soft-agar in a 96-well plate and incubated at 37°C in 5% CO₂ with the indicated inhibitors. After 7 days, agar was solubilized and cells were lysed according to the manufacturer's instructions. Colony formation was quantified using the fluorescent cell stain CyQUANT GR Dye (Cell Biolabs Inc.) in the Synergy HT Plate reader using Gen5 software from BioTek Instruments Inc.

Cell migration (chemotaxis) was also evaluated by using a Boyden chamber (membrane pore size = 8 µm) (Cytoselect^®, Cell Biolabs, San Diego, CA, USA)^{65 (link)}. Rat primary pulp cells were seeded into the upper chamber in α-MEM containing 1% (v/v) FBS at a density of 2.0 × 10⁴ cells, and MMP-treated DMCs or incubated DMCs without MMP (0.01–1.0 µg/ml) as an experimental control in α-MEM containing 1% FBS were added to the lower chamber. After 2 h incubation, cells that passed through the membrane from the upper to lower chamber were removed by using cell detachment buffer (Cell Biolabs) for 30 min. Then, CyQuant^® GR dye (Cell Biolabs) was added to the lower chamber for fluorescent staining of migratory cells. Migration was quantified by using a microplate reader (ARVO MX, PerkinElmer, Waltham, MA, USA) at 485 nm/535 nm fluorescence intensity (n = 8 for each group). Cells not treated with DMCs in the lower chamber served as a negative control. Each MMP molecule incubated alone (0.01–1 µg/ml) was used as an additional control.

We used the Cytoselect 96-well Cell Transformation Assay (Cell Biolabs, San Diego, CA, USA) to assess colony formation according to the manufacturer’s instructions. We counted live cells (variability >95%) using the Countess automated cell counter (Invitrogen, Waltham, MA), and each cell line was counted twice. The cells were seeded in soft agar at a density of 1 × 10 ⁴ cells per well, and duplicate plates were tested using Quantitation of Anchorage-Independent Growth and colony formation. The colony formation was quantified by solubilizing the soft agar, lysing the cells, and incubating cell lysates with the CyQUANT GR Dye (Cell Biolabs) with the GLOMAX Multi Detection System (Promega, Madison, WI, USA). In addition, the colonies were imaged under a microscope (Zeiss Axio, Carl Zeiss, Oberkochen, Germany).

To assess colony formation, the CytoSelect 96-Well Cell Transformation assay (Cell Biolabs, San Diego, CA, USA) was used according to the manufacturer’s instruction. Briefly, PCa cells were seeded in soft agar at 2500 cells/well. After 7 days of incubation at 37 °C in 5% CO₂, colony formation was quantified by solubilizing soft agar, lysing cells, and incubating cell lysates with the CyQUANT GR Dye (Cell Biolabs, San Diego, CA, USA) followed by analysis.

Trypan blue, soft agar growth, and MTT assays were used to determine growth and survival. For trypan blue assay, cells were plated at a density of 1 × 10⁴ cells/well in 24-well plates for 2–3 days, and then trypsinized, combined with the Trypan blue reagent (Sigma, St. Louis, MO, USA), and cell numbers were counted. For soft agar assay, cells were seeded at a density of 10,000 cells/well in 96 well plate containing semisolid agar media. Transformation ability of these cells was measured using CytoSelect^TM 96-well Cell Transformation Assay (Cell Biolabs, San Diego, CA, USA) following 6–8 days incubation. Fluorescence was read on SpectraMax M5 plate reader (Molecular Devices, San Jose, CA, USA) using 485/520 nm. Briefly, cells growing in semisolid agar media were solubilized, lysed, and incubated with CyQuant GR Dye (Cell Biolabs) for measuring fluorescence. For cell proliferation, cells were seeded in triplicate at a density of 4 × 10³ per well in 96-well plate. Cell proliferation was detected following 72 h incubation essentially as described previously [20 (link)] by measuring absorbance at 570/650 nm.