Hi all,
I had been using ThermoFisher's mouse T-activator kit (Catalog number: 11456D) to expand CD8 T-cells from mouse splenocytes. For around 2 years I had great results, got 200-fold expansion every time.
However, since November, this product has completely stopped working for me. I've tried replacing every reagent: new batches of media, FBS, IL-2, and different lots of the Dynabeads themselves. I've even gotten new mice and tried different mouse strains. No rescue of that 200-fold expansion that I had gotten used to seeing.
I've contacted their tech support to see if they've made any changes to the manufacturing process at all and have been told there has not been.
I realize there are other products (I've already had a chat with our Miltenyi rep) but I'm trying to get a last (hopefully) experiment done to add to a manuscript before it gets submitted. All the previous experiments for the paper were done with the ThermoFisher beads and I anticipate it would be a bit awkward to switch methods right at the end.
Does anybody have any insights? Has anybody else experienced a similar drop-off in quality with this product? I'm trying to figure out if there's something in-house I can do to try and fix this or if this is something on the manufacturer's end
Did you batch test your serum (fbs) and reserve it at the start of the assay? For my t cell cultures there was massive differences in the t cell growth in response to stimulation depending on the batch of serum. We used a company who sent us batches of serum for testing before we purchased 50 bottles that they stored for us and sent out on request. I never bought off the shelf FBS for stimulation and proliferation assays, it's fine for cell culture maintenance but can be massively variable for stimulation assays - lucky if you get a good batch, also always aliquoted and kept FBS frozen, in fact I generally made a massive 2×strength batch of supplemented culture media and froze it down in appropriate sized aliquot, then used that as my culture base adding the appropriate drugs/cytokine etc to make 1x media on the day. Certain stimulants eg LPS will also vary hugely by batch, but not sure if you're using any other stimulants or feeder factors/conditioned media. Good luck!
Hey Amanda, great suggestions. In my troubleshooting I tried using multiple FBS batches and also serum-free media that is supposedly well-defined for T-cell culture. No improvement. It could very well be that the one batch that we had for that year or two was the rare, ideal batch and most batches are not well suited for this protocol. If that's the case, then it's probably just best to explore other methods
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