Click-iT® Plus Chemistry
The copper concentrations typically used in traditional click chemistry reactions can affect fluorophores such as green fluorescent protein, mCherry, and R-phycoerythrin.
Click-iT® Plus EdU may be effectively employed in a low-copper reaction, enabling DNA synthesis–based cell proliferation assays that are compatible with multiplex imaging or flow cytometry experiments.
Biomolecules exhibit varying sensitivity to copper concentration. The high copper concentration used to catalyze traditional click reactions maintains the fluorescence of APC, but not GFP. At medium copper concentrations, GFP and APC fluorescence is maintained, but phalloidin binding is lost (Table 1). With Click-iT® Plus reagents, the copper concentration can be optimized to preserve the signal in your fluorescent proteins and also drive the EdU click reaction.
Table 1. The effects of copper on fluorescence and phalloidin binding.
Fluorescence and phalloidin binding maintained?
Click-iT® Plus EdU vs. traditional click reactions
- Faster than traditional BrdU
- Brighter than traditional BrdU
- Minimal cell disruption
- Compatible with GFP multiplexing
- Detection strategies for imaging and flow cytometry
When antibody-based BrdU detection is compared to click chemistry–based EdU detection, the HCl denaturation required for BrdU results in loss of signal from GFP. Traditional click chemistry generates a brighter signal than antibody-based BrdU, but the copper catalyst concentration results in loss of GFP signal. The low-copper reaction with Click-iT® Plus EdU results in bright EdU signals and retains the fluorescent signal from GFP (Figure 1). In addition, detection of EdU with the Click-iT® Plus Alexa Fluor® 488 picolyl azide was compatible with PE-Cy® 7 fluorescence in a flow cytometric assay for cell proliferation (Figure 2).
Figure 1. Cell proliferation assays using A375 melanoma cells expressing Erk2-GFP comparing traditional antibody-based BrDU (A), Click-iT® EdU (B), and Click-iT® Plus EdU (C).
Figure 2. Compatibility of Click-iT® Plus EdU reaction with R-PE tandem conjugate (R-PE Cy®7) fluorescence.
Direct substitution with improved results
Click-iT® Plus EdU can substitute directly for your traditional Click-iT® EdU assay in either flow cytometry or imaging applications (Table 2).
Table 2. Replacing existing Click-iT® kits with Click-iT® Plus kits.
|If you are using||Replace with|
|C10337||Click-iT® EdU Alexa Fluor® 488 Imaging Kit||C10637||Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit|
|C10338||Click-iT® EdU Alexa Fluor® 555 Imaging Kit||C10638||Click-iT® Plus EdU Alexa Fluor® 555 Imaging Kit|
|C10338||Click-iT® EdU Alexa Fluor® 594 Imaging Kit||C10638||Click-iT® Plus EdU Alexa Fluor® 594 Imaging Kit|
|C10340||Click-iT® EdU Alexa Fluor® 647 Imaging Kit||C10640||Click-iT® Plus EdU Alexa Fluor® 647 Imaging Kit|
|Flow cytometry applications|
|C10419||Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit, 50 tests||C10634||Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit, 50 tests|
|C10420||Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit, 50 tests||C10632||Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit, 50 tests|
|C10424||Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit, 100 tests||C10635||Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit, 100 tests|
|C10425||Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit, 100 tests||C10633||Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit, 100 tests|
BioProbes® Journal articles