A 4× stock solution of 40 µg/mL PI and 0.4% Triton X-100 in PBS was made immediately following transfer of cells to a 96-well plate. A multi-channel pipet (Cat. no. EW-24553-07; Eppendorf South Pacific, North Ryde, NSW, Australia) was used to dispense 50 µL stock solution to each well containing 150 µL cell suspension, giving a final working concentration of 10 µg/mL PI and 0.1% Triton X-100. PI fluorescence based on binding of dsDNA was measured using a SpectraMax M5 fluorometer (Molecular Devices, Sunnyvale, CA, USA) at an excitation of 485 nm, emission of 612 nm, and a cutoff of 610 nm. Cells were incubated at room temperature for 1 h with measurements made from the top of the plate every 5 min. Blanks contained 150 µL PBS and 50 µL 4× stock solution. Data was analyzed and standard curves generated using SoftMax Pro v5.01 (Molecular Devices).Plating hESC at varying densities
MEL1 and MEL2 cells were dissociated into clumps as previously outlined and a cell count performed on triplicate 150-µL aliquots of each cell line using the Spectramax fluorometer. PI fluorescence signals obtained were used to estimate cell number using the standard curve and equation y = 7 × 10−4x. Cells were plated in 12-well plates containing glass coverslips coated with Matrigel (34.7 µg/ cm2; BD, Franklin Lakes, NJ, USA) at densities ranging 0.05-2 × 105 cells/well and cultured for 10 days. mTESR medium (Stem Cell Technologies, Melbourne, Australia) was changed daily.Evaluation of hESC plurpotency markers after different seeding densities
MEL1 and MEL2 were analyzed by observation of the pluripotency markers GCTM2 and CD9 using immunofluorescence and flow cytometry. For immunofluorescence, cells were fixed with cold ethanol for 30 min. After each step, cells were washed twice in TRIS-buffered saline with 0.05% Tween-20. Cells were permeabilized with 0.1% Triton X-100 then blocked for 15 min in KOSR. Cells were stained with CD9 antibody (2.5 µg/mL; The Australian Stem Cell Centre, Melbourne, Australia) for 30 min and then anti-mouse IgG Alexafluor 488 (1:2000, 1 µg/mL) in the dark for 30 min, counterstained with 0.1 µg/mL Dapi, and mounted with anti-fade solution. A mouse IgG isotype control was used to determine nonspecific background staining. Cells were visualized using an LSM 510 META on an AxioObserver using Z1 and Zen 2007 Light Edition SP1 software (Carl Zeiss, Jena, Germany). Flow cytometry for CD9 and GCTM2 (2.5 µg/mL) was performed on 106 cells/mL as previously described (12). Detection was carried out using AlexaFluor 633 anti-mouse IgM (GCTM2) and Alexafluor APC-750 anti-mouse IgG (CD9) (1:2000, 1 µg/mL final concentration, 106 cells/mL). Appropriate mouse IgM and IgG isotype controls were used to set background fluorescence levels. Data was analyzed using WEASEL Software V2.4 (Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia).Determination of end point cell numbers and growth curves
MEL2 cells were seeded in clumps at 0.5 × 105 cells/well in a 12-well plate. Dishes were coated with Geltrex (1:30 as per manufacturer's instructions) fibronectin (10 µg/cm2), or vitronectin (2 µg/cm2). Cultures were grown in StemPro media over 9–10 days with daily media changes. Triplicate Geltrex-coated wells were harvested at days 2, 5, 7, and 9 as clumps using collagenase IV (200 U/mL), or as single cells using TrypLE Express. Data was used to generate growth curves and population doubling times using a hemocytometer or the PI assay. Replicate extracellular matrix (ECM)–coated wells were harvested with collagenase IV on day 9 and cells were counted using the PI assay to determine end point numbers. An Incucyte cell imager was used in parallel (Essen Instruments, Ann Arbor, MI, USA), according to manufacturer's instructions.Results and discussion
To circumvent the time-and labor-intensive manual counting of hESCs prior to plating, we opted to use the DNA content of hESCs as a measure of total cell number. To first establish the relationship between hESC number and PI fluorescence, we seeded increasing amounts of manually counted single hESCs in microtiter plates and PI stained them in a one-step procedure through addition of a 4× stock solution of PI/Triton X-100 in PBS.
As shown in Figure 1A, there was a linear relationship between PI fluorescence and hESC number when using <2 × 105 cells per well of a microtiter plate. Since hESCs are often subcultured as clumps of varying sizes containing 20–100 cells, we next determined the relationship between PI fluorescence and hESC cell number in clumps. One half of an equal distribution of hESC clumps was next dissociated into single cells and manually counted while increasing amounts of hESC clumps were seeded into microtiter plates and PI-stained using the protocol described above. As shown in Figure 1B, the relationship between PI fluorescence and hESC cell number was found to be almost identical to that for single cells (linear slope y = 6 × 10−4x versus y = 7 × 10−4 x, where y is fluorescence and x is cell number), suggesting that PI had stained the DNA with equal efficiency in clumps as in single-cell suspensions. Standard deviations for the highest cell counts performed (2 × 105) on clumps were±30.6 fluorescence units equaling±44,000 cells/well of a 96-well plate, using the equation in Figure 1B. To avoid further error in back-calculating total cells in original culture conditions, the most accurate means of performing the assay was to maintain <2 × 105 cells within aliquots for analysis. This also allows for sufficient surplus cells for analysis or propagation.