How long to leave CD8+ t cells in culture before the are stimulated and expand for a T cell killing assay?

Actually, you should incubate the T cells with beads for a shorter period of time. Prolonged stimulus causes them to become anergic or undergo activation-induced cell death. I would limit co-culture with the beads to about 24 hours.

What kind of vessel are you culturing the cells in? After removing them from the stimulus, I find the cells tend to do best when you put them into U-bottom 96-well plates at an initial density of about 1 million cells/mL. So, in your case, if you're getting about a million cells after negative selection, you would briefly stim, remove the beads, adjust the cells to a volume of 1 mL, and split them across 5 wells of a U-bottom 96-well plate.

As for IL-2, be careful here. Every batch of IL-2 has its own activity as measured in bioactivity units (1 unit = the ED50 of metabolic activity in the cell line, CTLL-2). So there is not a single number you can use to convert between volume or mass and units. The best thing is to get the actual batch-specific bioactivity assay data from your supplier OR perform your own in-house assay to determine activity (I've linked an example protocol at the bottom of this post, but there's other variations of it that would work too). Once you have that number, you can determine how many ng/mL you need to add.

For mouse splenocytes, there IS such a thing as too much IL-2. If you have too much, you'll start inducing LAK cells, which will probably ruin your experiments. So it's worthwhile to be fairly precise. However, in general, if you have IL-2 at a final concentration in your culture at about 20-30 ng/mL, you'll probably be somewhere close to the optimal dose.

Final note on IL-2: it does not do well with freeze/thaw or prolonged storage at 4 degrees. You should make -20degC aliquots of your stock, and make these aliquots in very small amounts (200 ng per tube or something) so that you limit the freeze/thaw on each tube to 3-4 cycles.

Good luck.

Article Measurement of Human and Murine Interleukin 2 and Interleukin 4

Govinda Sharma

Also, I just noticed some other issues with your reagents:

1) Human T-activator won't work for mouse T cells. You need to get the version with anti-mouse CD3/C28.

2) Mouse T cells like to have beta-mercaptoethanol in the media (this is not required for human T cells so people often overlook this).

Christine Yougn

Thanks for this!

I am culturing my cells in 24 well plated after stimulation so I will try a 96 well.

Also I wrote that I use human beads but that was a typo. I use mouse beads.

I have read that people stimulate cells for up to 7 days or so... but it sounds like you wouldn't recommend this

I will give your suggestions a go! Fingers crossed!

Govinda Sharma

Try the 96 well U-bottom, not flat bottom

Dinesh Mohanraj

Hi all,

I have a question surrounding this. I am using a T-spot plate to culture my PBMCs (2.6*10^6 cells/ml) along with viral peptides. I harvest the supernatant from this plate and subject to surface staining with dr and 38. However, i have noticed very low dual expression of these markers, yet dr+ is preferrentially expressed in comparison to unstimulated. I am leaving them to culture for 20hrs. Is this too short of time for cell activation??

Thanks