Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Prior to the assay cells were either fixed in 70% ethanol and incubated with propidium iodide/Triton X-100 staining solution with RNase A (0.5ml PBS/BSA + 100U/ml RNase A + 0.5mg/ml propidium iodide

Upstream tips
- Prior to the assay cells were either fixed in 70% ethanol and incubated with propidium iodide/Triton X-100 staining solution with RNase A (0.5ml PBS/BSA + 100U/ml RNase A + 0.5mg/ml propidium iodide

Publication protocol

Normal primary laryngeal and hypopharyngeal SCC FaDu cells were exposed to porcine pepsin (0.01, 0.1 or 1mg/ml; Sigma-Aldrich, St. Louis, MO) at pH7 for 1 hour at 37°C, washed 3 times briefly in fresh media and incubated for a further 4, 24 or 36 hours at 37°C in complete growth media. To measure the effect of irreversibly inactivated pepsin on cell proliferation, pepsin was irreversibly inactivated by prior incubation at pH8 for 15 minutes at 37°C before decreasing the pH to 7 and incubating with cells. Cells were either fixed in 70% ethanol and incubated with propidium iodide/Triton X-100 staining solution with RNase A (0.5ml PBS/BSA + 100U/ml RNase A + 0.5mg/ml propidium iodide; Sigma, MO) and analyzed by flow cytometry 25, or, assessed for cell cycle distribution using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit below.

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