Cell Proliferation ELISA, BrdU

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 10000 cells/well were seeded. - Treated with Wnt3a ( 0, 10, 50, 100, 150, and 200 ng/mL) for 24, 48, and 72 h	- Cells were incubated for three hours at 37°C in 5% CO2 after addition of BrdU

Upstream tips
- 10000 cells/well were seeded. - Treated with Wnt3a ( 0, 10, 50, 100, 150, and 200 ng/mL) for 24, 48, and 72 h
Protocol tips
- Cells were incubated for three hours at 37°C in 5% CO2 after addition of BrdU

Publication protocol

MTT and BrdU assays were performed on 96-well plates. Ten thousand cells were seeded per well and cells were stimulated with escalating doses of Wnt3a (R&D Systems, Minneapolis, USA), namely, 0, 10, 50, 100, 150, and 200 ng/mL, for 24, 48, and 72 h. MTT (Sigma-Aldrich, St. Louis, USA) was dissolved in PBS at 5 mg/mL and 20 μL of MTT solution was added to each well followed by an incubation of three hours at 37°C in 5% CO2. Medium with MTT was then flicked out and 200 μL DMSO was added. To dissolve the crystals, cells were shaken for 15 min at room temperature. Plates were read out by an ELISA reader (Tecan Group Ltd., Männedorf, Switzerland) at 590 nm. For BrdU assay a commercial kit “Cell Proliferation ELISA BrdU colorimetric” (Roche, Mannheim, Germany) was used according to the manufacturer's instructions. After addition of BrdU labelling solution the cells were incubated for three hours at 37°C in 5% CO2. After the final washing step 100 μL/well substrate solution was added to the cells and the reaction was stopped after 15 min incubation at room temperature by using sulphuric acid. Plates were read out immediately by an ELISA reader at 450 nm. All assays were done in triplicate (n = 12). For calibration cells were seeded in different numbers (0,1 × 104 to 3 × 104).

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