Can I combine Click-iT or Click-iT Plus reactions with phalloidin conjugates used for actin staining?
We do not recommend using phalloidin conjugates for staining actin in combination with traditional Click-iT or Click-iT Plus reactions since phalloidin is extremely sensitive to the presence of copper.
For staining actin in combination with traditional Click-iT or Click-iT Plus reactions, we recommend using anti-α-actin antibodies for staining actin in the cytoskeleton. You can find a list of our actin antibodies here.
Another option would be to use the Click-iT Plus Alexa Fluor Picolyl Azide Toolkit (Cat. Nos. C10641, C10642, C10643). These Click-iT Plus toolkits provide Copper and Copper protectant separately which makes it easier to titrate the copper concentration to obtain optimal labeling with minimal copper-mediated damage. You may need to optimize the click reaction with the lowest possible concentration of copper and then perform the phalloidin staining.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
I am unable to analyze my cells stained with eFluor Organic Dyes today. What options do I have?
Our options will depend on the samples you are analyzing.
If cell viability is not critical, you can store your stained samples at 4 degrees C or on ice overnight in the dark and analyze the following day.
For samples stained with eFluor organic fluorochromes, we recommend that cells be suspended in 100 uL of Flow Cytometry Staining Buffer (Cat. No. 00-4222) and 100 uL of eBioscience IC Fixation Buffer (Cat. No. 00-8222); samples can be incubated for up to 3 days at 4 degrees C in the dark. Alternatively, the 1-step Fix/Lyse Solution (Cat. No. 00-5333) can be used. This is a great option when working with whole blood but also works for other cell types.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Can the eFluor Organic fluorochromes be used for intracellular staining?
Yes, the eFluor Organic fluorochromes can be used for intracellular staining. The eFluor organic fluorochromes maintain bright signal and require minimal changes in compensation when fixed with eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) and Permeabilization Buffer (Cat. No. 00-8333-56) or 1-step Fix/Lyse Solution (Cat. No. 00-5333-54, 00-5333-57) (as compared to live cells).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Is the eFluor 660 fluorochrome compatible with Anti-Cy5/Alexa Fluor 647 beads?
Yes, in-house studies have demonstrated that the eFluor 660 fluorochrome is recognized by Anti-Cy5/Alexa Fluor 647 beads. Side by side studies with Alexa Fluor 647 versus eFluor 660 conjugated antibodies have demonstrated comparable results.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
What buffers are compatible with the eFluor Organic fluorochromes and eVolve QDots?
The eFluor Organic fluorochromes and eVolve QDots can be used with flow staining buffers containing PBS and protein.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
