You don't need the BD software to analyse bead data. It's just based on fluoercence intensity after all. If you have FlowJo and Prism I can send you our protocol. It is also my understanding that you should be able to run Luminex beads on a flow cytometer without the need for the Luminex machine (though I've not personally tried this).

You don't need the BD software to analyse bead data. It's just based on fluoercence intensity after all. If you have FlowJo and Prism I can send you our protocol. It is also my understanding that you should be able to run Luminex beads on a flow cytometer without the need for the Luminex machine (though I've not personally tried this).

Thanks Ben. I have Flow Jo and Prism licence - so it would be great if you could send me your protocol. Wasn't sure about the Luminex test compatibility, with a flow cytometer: size of beads and how bead specificty would be assigned for a multiplex assay (since automatic on X-MAP system when you import LOT information).

Use Luminex.  There should be someone at your institution that has an instrument.

Haven't tried this yet, so can't recommend it, but ... FYI ... BioLegend just released a kit.

http://www.biolegend.com/legendplex

Thanks a lot Philippa. I was at a conference yesterday and just happened to meet our local representative for Biolegend. She said that the software is free. There is currently 50% off standard multiplex kits, which cover up to 13 analytes. They are still in the process of beta-testing over here, whilst they finalize their panels - so might be an opportunity to try the kits out for free.

I use Milliplex magnetic bead kits but do have a Luminex machine. I find them far easier to use than the CBA

Hi Burcu

I would recommend lysing the red cells first.

The cytokines will be relatively stable in the minus 80, but you should spin the media hard to ensure all the cells are removed before freezing - this ensures you measure the secreted cytokines, induced by stimulation and not the cytokines released on cell lysis.

I would also give consideration to storing the supernatants in a deep 96 well plate (1ml and 2ml capacity available) and creating daughter cells from this. This will mean repeat freeze thawing is not required and it also saves reformatting for any future ELISA assays. Having these samples stored safely, can greatly enhance your data series - you know quality will be good and you can add new tests later, if they are indicated in literature. 

Katrina

I suggest to use Biolegends  LEGENDplex products - worked perfect in my hands (though analyzing software did not recognized bead populations correctly - analyses were much easier using FlowJo software)

Actually, I used cell culture supernatants of antigen-secifically  re-stimulated T cells contained in murine splenocyte suspensions (that were lysed to get rid of red blood cells) 

Hi all, I plan to apply Biolegends  LEGENDplex products on serum of patients. Is it necessary to run samples in duplicates? Since the samples I have are around 300, it would be costly to run duplicates.But it seems that some paper of high quality do that. Is duplicates on the must-do list?

Hi Rongguang,

Personally, I would suggest to run the samples in duplicates. You can cut reagent volumes in half so you can analise the double of samples for the same prize.

Thank you Aina! That's a good suggestion.

I found running duplicates for some samples to prove the repeatability is also applicable when there are too many samples.