The 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay was used to quantify cell proliferation in encapsulated cells according to previous methods.[44 (link)] EdU, a thymidine analogue containing a terminal alkyne group that is incorporated into newly synthesized DNA, was added to each well at a final concentration of 1 μM for 10 h for days 1, 7, 14 and 21. To analyze the amount of newly synthesized DNA, a cell-permeable fluorescent azide was conjugated to the EdU alkyne using copper-catalyzed click chemistry. Briefly, the gels were fixed with 4% PFA for 30 min, rinsed 3X with DPBS, permeabilized with 0.5% Triton-X 100 for 30 min, rinsed, blocked using 5% milk for 2 h, and incubated with Alexa Fluor 488 conjugated azide (Invitrogen) diluted in 1M Tris (pH 8.5), 25 mM copper (II) sulfate and 0.25 M ascorbic acid. DAPI was used to stain nuclei. Constructs were imaged using a Zeiss 880 confocal microscope with a 25X oil immersion objective. EdU+ cells divided by total cells counted was used to depict proliferation in cells.
Alexa fluor 488 conjugated azide
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488 conjugated azide is a fluorescent dye that can be used to label biomolecules containing alkyne groups. It is designed for use in various biological applications, including cell imaging, protein labeling, and nucleic acid detection.
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3 protocols using alexa fluor 488 conjugated azide
1
Quantifying Cell Proliferation with EdU Assay
2
mRNA Labeling and Immunofluorescence Staining
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To label nascent mRNA, cells were pulsed and labeled as previously described (Jao and Salic, 2008 (link)), and following manufacturer’s protocols. Cells migrating in microfluidic devices were pulsed with 1 mM 5-ethynyl uridine (EU, Jena Bioscience) in complete DMEM for 4 h, and then fixed with 4% PFA in PBS for 30 min. After two PBS washes, the devices were carefully removed as described in the IF staining section. The cells were then permeabilized with 0.3% Triton X-100 (Sigma) in PBS for 15 min and incubated for 30 min with freshly prepared EU labeling buffer containing 100 mM Tris base (Sigma), 100 μM Alexa Fluor 488 conjugated-azide (Invitrogen), 4 mM CuSO4 (Sigma), and 100 mM ascorbic acid (Sigma) in water. After the incubation, cells were washed with PBS, and another round of 30 min incubation with EU labeling buffer and PBS washes was repeated. Cells were then incubated with primary antibodies and proceeded with the IF staining protocol.
3
EdU Incorporation Visualization
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EdU was added to the growth medium 2 h before fixing the cells, and EdU-labelled cells were visualized using the Alexa Fluor 488-conjugated azide (Invitrogen, A20012).
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