Soft agar assay was carried out in 6-well plates in triplicate for each cell line. An underlay of 2 ml media with 0.5% agar (2-Hydroxyethylagarose, Sigma A4018) was dispensed into each well and allowed to set. Cells were resuspended in media containing 0.4% agar and seeded at 5,000 cells per well in a volume of 1.5 ml. After 10–14 days, colonies were stained with iodonitrotetrazolium chloride (Sigma, I10406) and visualized with GELCOUNT imaging system (Oxford Optronix). Total colony number for each well was enumerated based on colony size and staining intensity.
Gelcount imaging system
Manufactured by Oxford Optronix
The GelCount imaging system is a lab equipment product designed for the automated analysis and quantification of gel-based assays. The core function of the GelCount is to capture and analyze digital images of gels, enabling accurate and reproducible measurements of various parameters such as band size, intensity, and molecular weight.
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4 protocols using gelcount imaging system
1
Soft Agar Assay for Anchorage-Independent Growth
2
Cisplatin and Radiation Effects on Colony Formation
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Colony forming assays were conducted by plating 200 cells per well in 12 well dishes. Six hours after seeding 1000X cisplatin (Calbiochem) solubilized in DMSO was added to a final concentration of 2 μM. Control plates and plates receiving radiation alone were treated with an equivalent volume of DMSO. Within 10 minutes of cisplatin addition, 4 Gy radiation was administered to the radiation alone or the cisplatin/radiation conditions. Plates were returned to a 37° incubator where colonies were allowed to grow for 6 days and subsequently fixed in 70% ethanol and stained with trypan blue in 10% ethanol. Colonies in triplicate wells were counted on a GelCount imaging system (OXFORD OPTRONIX) using identical settings.
3
Soft Agar Assay for Colony Formation
4
Prostate Cancer Cell Lines: Spheroid Formation and Drug Treatment
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The human prostate cancer LNCaP (ATCC® CRL-1740™) and DU-145 (ATCC® HTB-81D™) cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The 22Rv1 cells were a kind gift from Prof. Valentine Macaulay (University of Oxford). LNCaP and 22Rv1 cells were maintained in RPMI 1640 medium. DU-145 cells were grown in DMEM. All media were supplemented with 10% FBS (Sigma-Aldrich, Darmstadt, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany). Cells were kept at 37 °C under a 5% CO2. Cells were routinely tested for mycoplasma and found to be negative. For spheroid formation, 5000 cells were seeded in 200 μL of complete growth medium in round bottom, ultra-low attachment 96-well plates (#7007, Corning, New York, NY, USA). Spheroids were grown for 5–10 days, after which the 22Rv1 and DU-145 cells produced spheroids of approx. 400 μM in diameter, while the LNCaP cells yielded much larger spheroids (approx. 800 μM in diameter). The medium was renewed, and the spheroids were subsequently treated with the drug for 96 h. Spheroid size was monitored using the GelCount imaging system (Oxford Optronix).
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