CyQUANT® Cell Proliferation Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were plated in a 96 well plate at 2.5×103 cells per well	- Cells are incubated for 1–4 days.

Upstream tips
- Cells were plated in a 96 well plate at 2.5×103 cells per well
Protocol tips
- Cells are incubated for 1–4 days.

Publication protocol

Cell migration was measured using Corning transwell inserts (BD Biosciences) with 8.0 μm pore polycarbonate membrane. Membranes were coated with Collagen I (BD Biosciences) at 100 μg/ml. 1% FBS in DMEM was used in the lower wells as chemoattractant. Cells were trypsinized, trypsin inactivated with soybean trypsin inhibitor and washed in DMEM. 6×104 cells were added to the top transwell chamber and allowed to migrate for 4 hours. Cells were fixed and stained with Diff-Quik (Fisher Scientific) and a cotton swab used to remove non-migrated cells from the upper chamber. Migrated cells were counted in 3–5 fields/well with 2–3 wells/condition. Cells were used for experiments 48 hours after transfection. For invasion assays, BD BioCoat Matrigel Invasion Chambers, with 8.0 μm pore PET membrane in 24-well cell culture inserts (BD Biosciences) were used with 5% FBS as the chemoattractant. Cells were allowed to invade for 12 hours and were fixed, stained and counted as described above. For uPA inhibitor experiments, cells were treated with 0.1% DMSO vehicle, 10 μM amiloride or UK122 (EMD Millipore, Billerica, MA). In vitro cell number was measured using CyQUANT Cell Proliferation Assay kit (Life Technologies). Cells were plated in a 96 well plate at 2.5×103 cells per well and incubated for 1–4 days. Plates were frozen and processed together at the end of the experiment. Fluorescent signal correlated with cell number and was measured with 450 nm excitation and 520 nm emission filters.

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