BrdU Cell Proliferation Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-After cells were attached, they are treated without or with thyrostimulin and/or effector inhibitors for 24 h.	- Incubation time with BrdU was for 4h. - After addition of AlamarBlue, cells were incubated for 3 h at 37 °C.

Upstream tips
-After cells were attached, they are treated without or with thyrostimulin and/or effector inhibitors for 24 h.
Protocol tips
- Incubation time with BrdU was for 4h. - After addition of AlamarBlue, cells were incubated for 3 h at 37 °C.

Publication protocol

NIH:OVCAR-3 cells were trypsinized, counted, resuspended with FBS-containing media. For BrdU incorporation analysis, 1.25 × 105 NIH: OVCAR-3 cells were plated into each well of 48-well plates and incubated for one day for attachment. Following attachment, the cells were washed, starved with serum-free medium and then treated without or with thyrostimulin and/or effector inhibitors for 24 h. 10 μM BrdU was added into medium for the last 4 h. To measure the amount of incorporated BrdU, BrdU cell proliferation assay kit (Cell Signaling Technology) was used following the manufacturer’s instructions. For long-term culture to observe the knockdown effect of TSHR on cell proliferation, the cells were resuspended with FBS-containing media and then seeded in 48-well plates (1500 cells/well). At indicated intervals, 10% medium volume of AlamarBlue (AbD Serotec) was directly added in the wells and the plates were incubated for 3 h at 37 °C. The fluorescent signal is monitored using an excitation wavelength at 560 nm and emission wavelength at 590 nm according to the manufacture’s protocols. All experiments were performed in a triplicate manner.

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Manufacturer protocol

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