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8.5
Remove rinse solution. 100 µL of 1% BSA in PBS is then given to the cells in
each well.
8.6
If performing antibody surface or intracellular labeling, proceed immediately to
step 9, otherwise continue to step 10.
9. Staining Intracellular or Surface Antigens (optional)
The PromoKine EdU-based Cell Proliferation Assay (HTS) can be used with antibodies
against surface and intracellular markers. To ensure the compatibility of your reagent or
antibody, please refer to Table 7.
9.1
Add antibodies against intracellular antigens or against surface antigens that use
RPE, PR-tandem or Quantum Dot antibody conjugates. Mix well.
9.2
Incubate the cells for the time and temperature required for antibody staining.
Protect from light.
9.3
Wash each well twice with 100 µL permeabilization solution. Remove the
solution. Add again 100 µL of 1% BSA in PBS to the cells.
9.4
Proceed with step 10 for analyzing the cells.
Fluorescent molecule
Organic dyes such as Fluorescein and Alexa dyes
PerCP, Allophycocyanin (APC) and APC-based tandems
R-phycoerythrin (R-PE) and R-PE based tandems
Quantum Dots
Fluorescent proteins (e.g. GFP)
Table 1: EdU detection dye compatibility.
* Compatibility indicates which involved components are unstable in the presence of copper catalyst for
the EdU detection reaction (either the fluorescent dye itself or the detection method). Not all GFP
antibodies recognize the same antigen site. Rabbit and chicken anti-GFP antibodies result in a good
fluorescent amount. The mouse monoclonal antibodies tested are not recommended for this application
because they do not generate an acceptable amount of fluorescence.
10. Imaging and analysis
10.1
Close the 96 well plate by using a sealing film, if desired.
10.2
Fluorescence is quantified by scanning the plate using an automated imaging
platform equipped with filters appropriate for the dye used. Images of each well
can be taken by microscopy.
The Excitation and emission maxima of the available dyes are listed in table 8.
Instruction Manual
Compatibility
Compatible
Compatible
Use R-PE and R-PE based tandems
after the EdU detection reaction
Use Quantum Dots after the EdU
detection reaction
Use anti-GFP antibodies* before the
EdU detection reaction or use organic
dye-based reagents for protein
expression detection
9
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