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These harsh treatments can adversely affect cell morphology and antigen recognition sites, as well as image quality.
The denaturants also limit the ability of the Brd U assay to be multiplexed with fluorescent proteins (e.g., GFP, RFP, m Cherry) or phycobiliproteins (e.g., R-PE, R-PE tandems), which are regularly used in imaging or flow cytometry applications.
Erk2-GFP–expressing A375 melanoma cells were transduced with Cell Light® Talin-RFP (orange) overnight, and then pulsed with 10 µM Ed U for 2 hr and labeled using the Click-i T® Plus Ed U Alexa Fluor® 647 Imaging Kit (pink) and Hoechst® 33342 nucleic acid stain (blue).
Coverslips were mounted with Pro Long® Gold Antifade Mountant and imaged using a Nikon® ECLIPSE® E800 microscope with Semrock® DAPI, FITC, TRITC, and Cy®5 optical filter sets.
To make full use of such information, it is important to correlate “cell types” to gene expression.
Toward this end, we have developed highly sensitive method of fluorescent dual-probe ISH, which is essential to distinguish two cell types expressing distinct marker genes.
The click reaction and subsequent wash steps are typically completed in 60 minutes, and newly synthesized DNA can be detected and quantified using image-based techniques or flow cytometry. GFP and RFP compatibility with the Click-i T® Plus Ed U assay.Although it eliminates the complications of working with radioactivity, the Brd U proliferation assay is both difficult to perform consistently and time consuming, typically requiring 6–24 hr to complete.In the standard Brd U assay, cells are incubated with Brd U and then treated with acid, heat, or enzymes to denature the DNA and facilitate detection of the incorporated Brd U molecules by anti-Brd U antibodies (Figure 2A).The Click‑i T® Plus Ed U assay represents a significant breakthrough in the evolution of cell proliferation measurements (Figure 1).
The most accurate cell proliferation assays directly quantitate newly synthesized DNA by following the incorporation of a deoxyribonucleoside analog that contains a detectable tag.
(B) The small size of the Ed U detection reagent, Alexa Fluor® picolyl azide, eliminates the need to denature the DNA for the detection reagent to access the nucleotide. Detection of cell proliferation and GFP fluorescence in mouse tissue.