Can I use homemade fixation buffers (or other commercially available fixatives) on my cells after staining with eVolve-conjugated antibodies?
No. For optimal results, we recommend that you use the IC Fixation Buffer (Cat. No. 00-8222-49) or 1-step Fix/Lyse Solution (Cat. No. 00-5333-54 or 00-5333-57). Impurities in fixation buffers can severely affect fluorochrome brightness.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Will eVolve fluorophores survive fixation?
Absolutely. eVolve fluorophores retain their fluorescent properties after fixation with eBioscience's fixation buffers such as IC Fixation Buffer (Cat. No. 00-8222-49).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Can I use homemade fixation buffers (or other commercially available fixative) on my cells followed by flow cytometric analysis?
No. We do not recommend the use of other fixatives. We validate our studies using eBioscience's IC Fixation Buffer (Cat. Nos. 00-8222-49, 00-8222-52) and 1-step Fix/Lyse Solution (Cat. Nos. 00-5333-54, 00-5333-57). Other recipes or manufacturer's products may give inferior results.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
I can not run my samples for flow cytometric analysis immediately. What do I do to preserve the best intracellular staining?
For optimal results, we recommend that you analyze stained cells immediately.
However, if you do need to store your samples that were stained with antibodies conjugated to organic fluorochromes, we recommend you complete your staining protocol and fix your samples with IC Fixation Buffer (Cat. Nos. 00-8222-49, 00-8222-52) (100 µL sample with 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (Cat. Nos. 00-5333-54, 00-5333-57). Cells can be stored in these buffers for up to 3 days in the dark at 4 degrees C.
We have observed minimal impact on brightness or FRET efficiency/compensation when using the IC Fixation (Cat. Nos. 00-8222-49, 00-8222-52) or 1-step Fix/Lyse Solution (Cat. Nos. 00-5333-54, 00-5333-57). Differences in fixation buffer quality can affect fluorochrome brightness or FRET efficiency. Fixation of tandem dyes, such as APC-eFluor 780 and PE-Cy7, does not increase the amount of compensation required from the APC or PE detector, respectively. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Can samples stained with antibodies conjugates to tandem dyes (i.e., APC-eFluor 780, PE-Cy7, etc.) be fixed and stored?
Yes, samples that are stained with tandem dye conjugated antibodies can be stored in IC Fixation Buffer (Cat. No. 00-8222-49, 00-8222-52) (100 µL sample with 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (Cat. No. 00-5333-54, 00-5333-57) for up to 3 days in the dark at 4 degrees C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
