Please could anyone say when you use tandem dyes (PerCP-Cy5.5 or APC-Cy7 or others) what buffer solution do you keep your cells in if you can't analyze them immediately? Thanks in advance!
If you fix them with PFA or a fixation buffer (BioLegend or BD): keep 1-2 days (I wouldn't do more than 3), in the dark, at 4*C. The tandems will be fine.
If you can't fix them -- keep until the next morning, then run them.
Keep it in something with FCS, and preferable EDTA (usual components of FACS buffer). Don't keep it in fixative -- fix cells for 5 min, and resuspend in something like 200uL of FACS buffer.
FACS buffer: PBS, EDTA (10%), FCS (5%).
PerCP/Cy5.5 is probably the most stable tandem of all -- it's pretty hardy. PE/Cy7 not as much, but it's fine. Just keep it out the light and don't keep the cells in fixative longer than you have to.
Dear Yuliya, Do not keep the cells. Analyze them immediately. Otherwise, you will obtain compromised results.
I find them quite stable actually. At some point I analyzed cells stained with Pe-Cy7 a week after staining and it looked almost as bright as when I measure immediately. In that case I kept the cells in the eBio 1xPerm solution from the Foxp3 staining buffer kit. You should of course keep them cold and in darkness to prevent dissociation of the tandem dye and bleaching. As a buffer, PBS/BSA or PBS/FCS should do the job, but as I said, even a detergent-containing buffer didn't compromise my staining.
Nevertheless, you should test the stability of your staining for your specific settings!
Brightness will not be a proble but tandem dye dissociation and chenges in spectral overlap. In multi-color assay it;s beter to store the sample overnight than the stained sample
As always, depending on sample and AB respective dye.
We have the policy: activation markers as soon as possible, stable markers over night. Storing at 4°C. When ever possible, we do a pre validation of storage fixing etc. If pretesting with fixation reveals good results, this is a good oppertunity to hold the samples after stating for several days ( in our setting at 4°C, be careful, some fridges may have only "one way thermostats", which results in freezing peaks well below 0°C (and thus destroying the cells, even then fixed).
If not tested, tandem conjugates are extremely unstable, when stored as a master mix (in multi color setting) for a longer time.
In my opinion the time to the measurements depends also 1) on the kind of measurement and 2) the kind of antigen that is stained.
1) If receptor densities on the cell's surface is of interest than it is important to measure the sample as soon as possible due to loss of antibodies bound to their antigen.
2) If cell populations should be determined (for example T-cells) than you can store the formaldehyde fixed samples at 4°C overnight in PBS. Usually you get equal distribution of cell populations compared to a sample measured directly after staining. But the separation of cell populations can be slightly impaired to my experience.
I hope my anwer will help.
Best regards
Dirk
Thank you so much! We use CD56 PerCP Cy5.5, so I hope it's ok to leave stained cells overnight.
If you fix them with PFA or a fixation buffer (BioLegend or BD): keep 1-2 days (I wouldn't do more than 3), in the dark, at 4*C. The tandems will be fine.
If you can't fix them -- keep until the next morning, then run them.
Keep it in something with FCS, and preferable EDTA (usual components of FACS buffer). Don't keep it in fixative -- fix cells for 5 min, and resuspend in something like 200uL of FACS buffer.
FACS buffer: PBS, EDTA (10%), FCS (5%).
PerCP/Cy5.5 is probably the most stable tandem of all -- it's pretty hardy. PE/Cy7 not as much, but it's fine. Just keep it out the light and don't keep the cells in fixative longer than you have to.
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