Figure 7 shows co-labeling of Jurkat cells with Click-i T® Plus Alexa Fluor® 488 picolyl azide to detect incorporated Ed U and with PE-Cy®7–conjugated mouse anti–human CD4 antibody.
This dual-parameter flow cytometry experiment demonstrates the preservation of the phycobiliprotein fluorescence as well as of the antigen recognition sites on the primary antibody. Dual-parameter plot of fluorescence of cells labeled with the Click-i T® Plus Ed U Alexa Fluor® 647 Flow Cytometry Assay Kit and GFP.
Both cell samples were treated with Hoechst® 33342 nucleic acid stain (blue); high-content analysis was performed using the Thermo Scientific® Cellomics® Arrray Scan® VTI HCS Reader.
The ability to multiplex Click-i T® Plus Ed U assays with other fluorescent probes opens the door to a more complete analysis of cell function.
The harsh treatment required for the antibody-based Brd U assay resulted in the loss of the GFP signal, as seen by the absence of green fluorescence in Figures 4A and 4B.
The proliferation signal from A375 melanoma cells expressing an Erk2- GFP fusion was detected using either Brd U or Click-i T® Plus Ed U (Figure 4).
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).
These harsh treatments can adversely affect cell morphology and antigen recognition sites, as well as image quality.
(A) Without DNA denaturation, Brd U is inaccessible to antibodies used for detection.
(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.Constitutively expressed β-actin–GFP fusion (green) is seen in the smooth muscle band below the bright Click-i T® Plus Ed U–labeled proliferating cells (red) of the intestinal villi; the tissue was counterstained with DAPI nucleic acid stain (blue).