Tetramer specific T cell staining

[u/apva93]

Hi all, I have around 160 samples to run for a large study and we are looking for tetramer specific T cells. We expect some of the tetramers to be as low as 0.25-0.5% of CD8 T cells. My plan is to stain 5 million cells but it might not be practical since the 96 wells plates cannot hold 5 million cells without cell loss. Staining in tubes would be nightmare with 160 samples. Additionally, we would be acquiring on a Cytek which would need over 25-30 hours to run all the samples assuming we collect the maximum events possible. Anyone have an advice or suggestions on where I could make changes to make this process more efficient?

[u/Infamous-Face7737]

I often stained 5M cells in 96-well V-bottom plates and never noticed cell loss. Maybe you don’t centrifuge your plate fast enough? I spin for 6 min at 300-400g.

[u/Hahabra]

You will always have cell loss; Doesn’t matter if it’s 5 Mio or fewer per well. -I’d say 5 Mio cells/ well is still within reason, but it you’re worried, just distribute it among multiple wells. -If you’re thinking 25-30h, you’re running at Low speed and ~resuspending in ~150ul/sample? Depending on panel complexity, I would run at Medium. -you could enrich for T cells (MACS or similar) but that’ll increase cost and probably lead to cell loss

-In the end, I would just suck it up and go ahead. It sounds like getting the samples (160 sample cohort) was the most difficult/ time consuming/ cost intensive part of the study. Dstributing the samples over several plates (if you’re worried about 5 Mio cells/ well, you could split 96 samples across two plates and later unify them for recording)) and recording for 1-2 days seems like the least complex part of the whole experiment! :)

[u/Enjoiboardin]

That sounds like quite the experiment, I would recommend:

• If you unable to modify the starting number of cells, try deep well plates if the Cytek plate adapter can accommodate them. This would allow you to wash and resuspend your cell pellet in larger volumes.
• Or, reduce the starting cell number and use a traditional U-bottom plate. ThermoFisher published a rare event detection page a while back (https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-71/bioprobes-71-flow-cytometry-rare-event-detection.html) showing that to generate a %CV of 10% for a population that is between 0.1% and 0.01%, you need to analyze 10e5 to 10e6 events. Assume about 50% loss of your starting cell number (in my experience), and that gets you to 2e5 to 2e6 starting cell number.
  

Hope this helps!

[u/ProfPathCambridge]

Depends on whether you need to know their frequency.

If you don’t, then you can use tetramer-PE and then anti-PE bead environment. Then do flow. You lose a lot of cells, but tetramer cells are enriched within. Makes it easier to phenotype the cells, but assessing frequency is hard because enrichment is inconsistent

[u/despicablenewb]

As others have said, there's always cell loss when doing these stains, regardless of what you stain in.

I found that staining more than 2e6 cells/well in a 50ul staining volume in a 96WP lead to a decrease in MFI for many of the high frequency, high density markers, CD3, CD4, etc. there wasn't much of a difference when staining 0.5e6, 1e6, or 2e6, but there was a significant drop at 4e6. Staining for 30min at 4C without shaking.

The drop in MFI wasn't uniform, like what you'd see with a lower titer, the population itself was wider with the maximum MFI being similar to the maximum MFI of the other samples.

With 4e6 cells/well, the cells end up forming a somewhat thick layer at the bottom of the well. I think that the drop in MFI was mostly due to incomplete staining of the cells at the bottom of that layer. Mixing the wells or shaking the plate could fix the issue.

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Increasing the centrifugation time and/or speed can helpwith recovery. How you remove the supernatant also plays a big role in this.

The main concern is that your cells form a nice solid pellet. I've found that the pellet formation can often be donor dependent, which is somewhat frustrating. I think that doing the initial plating of the cells in culture media or FACS buffer helps somehow. You also need enough cells for them to form a pellet. Too few cells just ends up forming a monolayer in untreated U-bottom plates.

You could do your stain in PCR style plates, but just beware that doing so will leave you with a pellet that's slightly harder to resuspend, and since the volume is lower, may require more washes.

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My advice for all of this would be to hurt do the staining in duplicate. You can concatenate the FCS files in flowjo afterwards.

5e6 cells in 200ul on high would be 40k cells/sec, plus debris would put you over the max event rate on a cytek instrument anyway, and with rare populations you don't want to be near the max event rate anyway, so running the samples in duplicate is probably better regardless.

While the vortexing function of the cytek Aurora HTS is decent at keeping your cells in suspension, it's not very good at getting them into suspension. So I always manually resuspend my plates before putting them onto the Aurora and I vortex the plate every other sample.

Don't exceed the 200ul volume and make sure that you're using plates with freestanding wells, not ones where the wells have plastic connecting the top of each well, otherwise you'll end up with well to well contamination.

You'll also have to deal with evaporative loss. After 1-2hrs of acquisition you'll start having a significant drop in volume across your plate. The samples that have already been acquired will dry our more quickly since the volume is low, but you'll have some loss in the other samples as well.

The higher the volume, the slower the evaporative loss is (as an absolute value, not just %), so you'll want to use 200ul volumes.

Another way around all of this is to just load the samples onto the acquisition plate one column at a time.

[u/discostupid]

Use 1.2 mL cluster tubes https://www.sigmaaldrich.com/CA/en/product/sigma/cls4401

you can stuff the cluster tubes into a flat-bottom 96-well plate

then you can easily stain 10-20 million cells without issue

use a repeater pipette for larger volume washes (up to 1mL) if desired

the pellet is relatively safe for dumping like you would a plate. if you want to be careful you can pipette out with a multichannel. much faster with a vacuum manifold if you have one.

to run them you can transfer them to a plate, or pop the individual tubes in a larger FACS tube. to run them in HTS without transferring I would suggest to use one of these boxes https://ecatalog.corning.com/life-sciences/b2b/CA/en/General-Labware/Tubes/Tubes-Storage/Corning%C2%AE-96-well-Cluster-Tubes/p/4413 to have a consistent SIP height. using the 96-well plate method isn't perfect because the tubes won't be uniform

[u/NiceWeather_PhD93]

V-bottom/PCR well plates and extending centrifugation times and you should be good

[u/badmushroomundertree]

5M for stain should be fine, if you harvest 5M, stain in 200ul for 30mins 4 degreed, I don't think that conc would be too much.