Scepter handheld cell counter

100 µl cell suspension of SKNAS (1 × 10⁴ cells/well) was seeded in 96-well culture plates (Corning Incorporated). After culturing to 80% confluence the supernatant was removed and transfection media was added to the cells. 48 h post transfection, cells were counted using a 60 µm sensor for the Scepter handheld cell counter (Millipore) [26 ]. Cell proliferation was measured using the MTS/MPS Cell Titer 96^® One solution Reagent (Promega) and detecting the color variation (FLUOstar Omega, BMG Labtech) as per the manufacturer’s recommendations. The absorbance values were normalized to the mock transfection and expressed as a percentage. All experiments were repeated three times. Cell cycle analysis was performed using the Cell-clock cell cycle assay (Biocolor). Images were subsequently analyzed using Image J image analysis as per the manufacturer’s instructions. The data presented is the average of three biological replicates. Each experiment series was repeated in triplicate.

Cells grown in either DMEM 10% FCS or PPRF-msc6 with different albumins were used to measure colony forming ability. Cells were harvested and counted using a Scepter hand-held cell counter (Millipore) and a total of 100 cells plated in triplicate into α-MEM (Life Tech), 10% fetal calf serum (Moregate Serum, New Zealand) medium in a p100 culture dish (Falcon). 14 days later cells were fixed with methanol and stained using 0.1% crystal violet and colonies counted.

100 μl cell suspension of SKNAS and NB69 (1 × 10⁴ cells/well) was seeded in 96-well culture plates (Corning Incorporated). After culturing to 80% confluence the supernatant was removed and transfection media was added to the cells. 48 h post transfection, cells were counted using a 60 μm sensor for the Scepter handheld cell counter (Millipore) as per the manufactures instructions [29 ]. Cell proliferation was measured using the MTS/MPS Cell Titer 96® One solution Reagent (Promega) and detecting the color variation (FLUOstar Omega, BMG Labtech) as per the manufacturer’s recommendations. The absorbance values were normalized to the mock transfection and expressed as a percentage. All experiments were repeated three times.
Cell cycle analysis was performed using the Cell-clock cell cycle assay (Biocolor). Images were subsequently analyzed using Image J image analysis as per the manufacturer’s instructions. The data presented is the average of three biological replicates. Each experiment series was repeated in triplicate.