Following siRNA knockdown, TRF1-FokI U2OS cells were induced with doxycycline (40ng/μL1) and synchronized in G2 with RO-3306 (Sigma) (10μM) 24hrs before harvest, followed by tamoxifen (4-OHT, Sigma) (1μM) and Shield1 Ligand (Takara Clontech) (1μM) 3hrs before harvest. Cells were pulsed with EdU (10μM) 1hr before harvest. Cells on glass coverslips were washed twice in PBS and fixed with 2% paraformaldehyde (PFA) for 10mins. Cells were permeabilized with 0.1% (w/v) sodium citrate and 0.1 % (v/v) Triton X-100 for 5mins. The Click-IT Plus EdU Cell Proliferation Kit with Alexa Flour 488 (Invitrogen) was used to detect EdU.
Click it plus edu cell proliferation kit with alexa flour 488
Manufactured by Thermo Fisher Scientific
The Click-IT Plus EdU Cell Proliferation Kit with Alexa Flour 488 is a tool used to detect and quantify cell proliferation. It utilizes the incorporation of the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) into DNA during active DNA synthesis. The incorporated EdU is then detected using a copper-catalyzed click reaction with Alexa Fluor 488 dye, allowing for fluorescent labeling and visualization of proliferating cells.
Automatically generated - may contain errors
3 protocols using click it plus edu cell proliferation kit with alexa flour 488
1
Synchronized TRF1-FokI U2OS Cell Imaging
2
Quantifying Telomere Replication in G2-Arrested Cells
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According to the experimental set up, cells were seeded on coverslips at a density of ~100,000 cells and treated with dye and light either prior or after G2 arrest. G2 arrest was carried out by treating cells with 7 μM CDK1i RO-3306 (Sigma) for 18–20 h. In the last hour of arrest/recovery cells were pulsed with 10 μM EdU. Coverslips were then harvested for FISH followed by EdU detection using the Click-IT Plus EdU Cell Proliferation Kit with Alexa Flour 488 (Invitrogen). Coverslips were then imaged and telomeres positive for EdU in non-S phase cells were quantified using the NIS Elements Analysis software.
3
EdU Incorporation Assay for Cell Proliferation
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Following transfection, cells were pulsed with EdU (10 μM) for 1 hr before harvest. Cells on glass coverslips were washed twice in PBS and fixed with 4% paraformaldehyde (PFA) for 10 min. Cells were permeabilized with 0.3% (v/v) Triton X-100 for 5 min. The Click-IT Plus EdU Cell Proliferation Kit with Alexa Flour 488 (Invitrogen) was used to detect EdU.
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