Evaluation of anchorage-independent proliferation in tumorigenic cells using the redox dye alamarBlue.

Cell proliferation is a key focus of research in cell biology, relating as it does to development, aging, immune activation, tumorigenesis and basic cellular processes such as signal transduction, DNA transcription and DNA replication. A wide variety of techniques for evaluating cell proliferation in culture have been described including direct cell counting by manual or automated means, cell cycle analysis by flow cytometry (8), incorporation of labeled or modified nucleotides into DNA (2,9,12), measurement of total DNA by DNA-binding fluorophores (3,13) and the detection of metabolically reduced dyes by absorbance or fluorescence spectroscopy (10,14). These various methods balance sensitivity, reliability, cost, instrumentation requirements and processibility in varying degrees, but all are directed at evaluating cells growing in suspension or as an attached monolayer. Proliferation relating to tumorigenesis may be best examined under conditions of anchorage-independent growth, with cells immobilized in soft agar or agarose (6). Evaluation of proliferation under these conditions has been limited to either direct manual counting of clonal colonies or automated image analysis. The former method is time-consuming and not conducive to the analysis of a large number of samples, whereas the latter requires expensive, specialized equipment. Here, we report the use of alamarBlue dye (Alamar Biosciences, Sacramento, CA, USA) to evaluate the proliferation of cells immobilized in agarose. AlamarBlue has previously been used to monitor proliferation in cells growing in suspension or as an attached monolayer (2,4,11). When added to culture medium, the dye is reduced by cellular mitochondrial enzymes, yielding a soluble product directly proportional to cell number. The product has a lower peak absorbance value and a substantially enhanced fluorescence (11), which can be measured spectroscopically in cell medium without further processing. Because the procedure does not require processing of either the culture medium or cells for the spectroscopic measurements, it is easily adaptable for use with cells immobilized in agarose or soft agar. Three tumorigenic cell lines were used in the experiments. EJ cells derived from a human bladder carcinoma (1,5) were maintained at 37°C under 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL) and 2 mM glutamine. NCI-H157 cells derived from a nonsmall cell lung carcinoma (CRL-5802; ATCC, Rockville, MD, USA) and SKOV3 cells derived from an ovarian adenocarcinoma (HTB 77 ATCC) were similarly maintained in RPMI-1640 medium supplemented as described above. Experiments were conducted using either 35-mm petri dishes or, in one case, 24-well plates. The petri dishes were prepared with an initial 1-mL underlay of 0.45% agarose in medium. Trypsinized cells were then mixed with a 0.25% agarose/medium solution at 40°C to a concentration of 5–20 × 106 cells/mL, and 1 mL of this cell mixture was added to each plate on top of the underlay. Following solidification of this cell-agarose layer, an overlay of 0.5 mL medium was placed over the agarose. Microwell plates (24-well) were prepared in a similar manner, except that the volume of the two agarose layers was 250 μL, and the volume of the medium overlay was 200 μL. Cells were grown for 14–21 days in a humidified incubator at 37°C under 5% CO2, with medium overlays replaced every 4 days. Proliferation was evaluated either by the microscopic counting of individual colonies (>200 μm) or by spectroscopy following addition of alamarBlue dye. For evaluations using the dye, the medium overlay was replaced with alamarBlue diluted in medium (1:2 for use with the 35-mm dishes and 1:3 for use with the 24-well plates), yielding an overall dye concentration for each plate of approximately 10% (vol/vol). The cells were incubated for 18–24 h at 37°C under 5% CO2 until obvious color changes indicated the presence of sufficient amounts of reduced dye for adequate quantitation. For the 35-mm dishes, 200-μL aliquots were removed from each plate at different time points, placed in a 96-well microwell plate and analyzed by light absorbance at 570 nm using 595 nm as the reference wavelength (manufacturer’s instructions). For the 24-well plate, fluorescence measurements were taken directly from the plate using a fluorescence plate reader with an excitation wavelength of 560 nm and an emission wavelength of 590 nm (manufacturer’s instructions). In one experiment, EJ cells were plated at varying concentrations, and after 2–3 weeks, they were examined for both colony counts and absorbance following addition of alamarBlue (Figure 1). Both manual colony counts and alamarBlue spectroscopy demonstrated equivalent increases as a function of cell plating concentration, indicating that the dye procedure provides as accurate a measurement of anchorageindependent proliferation as colony counts under these conditions. Similar results were obtained with two other tumorigenic human cell lines (H157 and SK-OV3) In a second series of experiments, immobilized EJ cells were grown in the presence of varying serum concentrations, a procedure that produces varying proliferation rates (7), and the relia-