Because I do Luminex assay in my lab, then I bring the plate to other place for reading. But I consider which step to stop.
Should I add assay buffer then bring there (with protect forom the light), Or I stop at wash buffer step then keep the sample in the wash bufer, after that I add assay buffer in other place before reading?
Can we leave the plate in wash buffer in 30 min like that? In theory , it should be ok, shouldn’t it ?
Thank you very much
Hello,
Are you doing the standard luminex protocol?
https://www.dartmouth.edu/~dartlab/uploads/Luminex%20Extracellular%20Protocol.pdf
Do you read the plate in assay buffer or wash buffer (I've seen both). Yes i'd transport in assay buffer, I would not want to leave plates in wash buffer for a prolonged time as it can affect the signal in similar assays like ELISA.
The other option you have is to use your transportation stage (30mins) as the Streptavidin stage and then add your final wash stage when you arrive? Technically the streptavidin stage should be on a shaker (and possibly incubated depending on beads), in the past with Luminex I have forgotten to use a shaker for this stage, and the results are still ok, but due to mixing, better results are achieved with a shaker.
Charlie
Hello
Thank you for your help.
The plate was read in assay buffer. And following my protocol, streptavidin stage is on a shake in 10 min
Today I keep the plate in assay buffer and transportation stage it. In my results, some samples can not be detected by machine. Even 2 the lowest dilutent standard in series are also low and undetected. I don't know because of during transport they lost fluorencence or the concentration of cytokine is low in the sample or technical skill. What do you think about the reason of undetection?
May be I should transport during streptavidin stage.
Thank you
Phuong
Hello,
Are you sure the Steptavidin stage is only for 10 mins- this seems rather short?
How many different cytokines were you doing? Were the bottom two standards low for all cytokine readouts or just certain cytokines? If they are all low and uniform, this could be a problem at any stage, detection not worked properly, or long enough, or one of the supplied reagents may be faulty.
How many ctyokines were you measuring, generally the more you measure and the more beads you use, the harder and more prone to mistakes the Luminex assay is (In my exp).
It is very possible as you stated that because you waited longer to read the plates, the bottom standards lost fluorescence, where as the more concentrated standards/samples were still usable.
Charlie
Hello
Thank you
For the streptavidin, I am sure. Only shaking in 10 min in protocol
Today, just 1 cytokine. But I will do others with around 20 chemokines tomorrow.
Only a group is low and few sample is high, over the range of detection.For the number of the bead and flourences of the bead, the machine tell ok. The time of transportation is around 20 min.
Thank you Charlie
Phuong
In my experience, the beads can be stored in PBS-T for quite a while. I have run a serology assay in my lab, re-concentrated the beads in PBS-T, travelled to a collegue 1hr drive away, and tested there, without any significant signal loss.
I guess a cytokine assay might be a bit more sensitive, but 30min in such a buffer might be ok. Otherwise, ask your supplier.
Good luck!
René
Hello,
The beads will be just fine in the final buffer for 30min. Just seal the plate and cover with foil to keep it dark. For longer than 30min you can put it in the fridge and it is still fine.
What is important, is to resuspend the beads before reading the plate. I read my Luminex plates in a different location too so I take a box of tips with me and a multichannel pipette - for mixing the beads by pipetting up and down a couple of times - do this just before reading the plate.
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