Cell loss during acquisition prep

[marthabrainard1]

Hi Cytoforum!

I know cell loss is expected during acquisition prep, specifically when washing with CAS, but does anyone have any suggestions to mitigate this loss? We recently have been losing more cells after our 2nd CAS wash (these cells were frozen in FBS + DMSO-no beads were added until we ran). Currently we prep cells by washing 2x in CSB and 2x in CAS (1500g, 3 min).

We were thinking of testing different spin speeds and longer centrifugation times (I know fluidigm recommends 800g for 5 min), but I wanted to see if anyone else has tested this.

[mleipold]

Hi Martha,

1500g seems really fast, even for fixed cells. We typically do 400-500g for unfixed cells and 600-800g for fixed cells. Usually 480g and 800g, respectively, for 5-10min.


Mike

[BjornZ]

Hi Martha,

I haven't used CAS specifically, but in 0.5% BSA in PBS, 800 x g for 2.5 minutes or 600 x g for 5 minutes give ~95% recovery in my experience.

"Recovery" is calculated from the number of cells left in the supernatant.
"High" and "Low" refer to starting concentration of cells (4,000/ul or 700/ul).
"+MeOH" and "-MeOH" refer to whether or not they were methanol permeabilized.
All of the spins were for 2.5 minutes, except the far-right one labeled 5 min.
Tested in triplicate in PBS/BSA-rinsed polypropylene PCR plates. Rinsing the plates with protein is a good way to improve recovery.

-Zach

[NikPaschal]

Hi Martha, Mike and Zach,

This is an extremely interesting topic for me and thank you all for bringing this up, I will share my experience as I am trying to get the maximum cell recovery for certain projects.
I have been running panels with extracellular and/or cytoplasmic markers and cell recovery was never the big issue. I would be in the range of 10-30% recovery starting with 3-4 x 10^6 cells to stain. On the contrary, I am having difficult times to obtain similar cell recovery with intranuclear staining. My target application here is to stain PBMCs (Treg focused marker panel) with extracellular, cytoplasmic, and nuclear targets.

So, these are my notes overall:

#1 Nuclear staining buffers
I have compared the FDM buffers for intranuclear staining with the ebio FOXP3 buffer set and the ebio gives me a better cell recovery (see attached pdf).
Is there a risk of “severe” signal loss from cadmiums with the ebio Foxp3 buffer set compared with the FDM buffers? – I haven’t tested this personally to be honest, but I have seen my comments in the forum on this issue.

#2 Washing steps
I have tried modifications to the FDM protocols (suggested in other posts) like single washing steps (without repetitions) and addition of cisplatin at the last 5 min of the extracellular staining step and got slightly improved cell recovery.

#3 Fixation
I am using the suggested 1.6% fresh fixation. My next testing attempt is to try 2% PFA (and 4%) instead of 1.6% (the suggested fresh fix from the Ampule).

#4 CAS
CAS wash seems to have a huge impact on the yield (see attached pdf). I would expect this step to be critical, but I feel that there is something more to this. My impression is that CAS solution is pretty much unavoidable because it is theoretically the cleanest (metal-contaminant wise) buffer you can get to wash your cells before acquisition, but I will give it a try with PBS/BSA.

To sum up, I feel that, at least in my hands, FDM intranuclear buffers are harsh enough that when combined with inadequate (fresh) fixation allow CAS solution to give the final blow-burst to the cells, leaving me with a poor (>>>10%) recovery.

Greetings to all from Athens, Greece.
Nikos

Nuclear_Antigen_Staining_Protocol_testing_CyTOF_BRFAA_NP_24102021.png (152.37 KiB) Viewed 3864 times

[jobagins]

Hi everyone

My very own personal experience, what I see very often peeps are doing, and what leads to truly bad recovery is simple yet devastating : doing staining in eppendorf tubes, or flow staining tubes.
I highly recommend switching to plate staining and *no flicking* to take out supernatant - gentle aspiration with multichannel pipette only. 400g before and 800g after fixation, 1,6% vs 2% PFA will not change anything.
Recovery is fantastic

Joanna

[marthabrainard1]

Thank you all for the responses! Nick, we've been testing the CD45 cadmiums and have seen ~70-75% barcoding efficiency with eBio nuclear staining kit. We haven't tested the cadmiums with the FDM buffers so I can't compare the two. We did incubate with eBio fix for 30 min and expose the cells to eBio fix/perm buffer, but I can't speak for signal in any other antibody channels as we didn't use a surface or intracellular mastermix in our early tests.


I'm sure you already know this, but if you are prepping cells for acquisition, we try and keep the cells we aren't immediately running, pelleted in CSB and only wash with CAS right before running
Martha

[cguidos]

Ho Joanna - can you give more detail about your plate-based stain/wash method?
1) What kind of plates - regular or deep-well?
2) If regular round or V-bottom? TC treated or flexible/soft?
3) washing details - what volume - how many?

Thanks

Cynthia

[mleipold]

Hi all,

Regarding PFA: I will generally agree with Joanna that 1.6% vs 2% PFA will often not make a difference. However, we did have a recent protocol where going from 1.6% to 2% did help. In this case, the residual buffer volume left after aspiration was enough to dilute the 1.6% to where we lost a bunch of cells (even when we were adding 4mL of 1.6% to a residual buffer volume of 100uL or so). In other words, the 1.6% added wasn't actually 1.6% as the final concentration. You might be able to get away with that with some gentle perms, but not the Methanol perm that is part of the protocol.

Simply increasing the PFA to 2% (initial concentration) gave us probably 30% increase in recovery.


Regarding plates vs tubes: I will generally agree with Joanna that I get a lot more reproducible staining and recovery in plates vs tubes. Even for barcoding experiments that result in a pool of several million cells, the steps up til pooling I do in plates. After pooling, I often switch to 15mL conical Falcon tubes; I try to avoid 5mL FACS tubes, as I have more trouble seeing the pellet and aspirating properly.

For several years, I've used polystyrene deepwell 1mL round-bottom plates for staining individual samples. These are the ones I use: Thermo Scientific™ Nunc™ 96 DeepWell™ Polystyrene Plates, item # 278605, Fisher cat# 12-565-552. The real advantage of these to me is that the polystyrene is clear/transparent: no matter what, I can *always* see my pellets in a way that I can't always seem the with polypropylene. For these, I typically do 500uL washes: it's a decent wash volume, but I don't have to worry about it splashing out of the well with the P1000 tip.

For some of our other projects that involve SmartTube WB or other things that require larger volume washes (~4mL), we've started using 48-well deepwell plates (technically 4.8-5mL volume, but we do 4mL washes to avoid splash-out with the tips). These are pointy/pyramid bottom, polypropylene (whitish), which can make the pellets harder to see at times, but as far as I know, there are no fully transparent 48-well deepwell plates.

The ones we use are like these: 48-Well Deep Well Plates, Axygen Scientific, Corning, Supplier # P-5ML-48-C, VWR cat# 12000-726. But we've also gotten similar plates from Agilent and some other suppliers.

These 48well plates are 8 rows by 6 columns. Each column is basically double-wide, so if you're using a multichannel, you'll want to go column by column rather than row by row (unless you rearrange your tip box).


Mike

[PaulineM]

Dear cyTOF community!

I’m re-opening this old thread because, as Nikos, I am experiencing a huge loss of cells during the last wash in CAS+, right before acquisition.

As I resuspend cells in more than 2ml of CAS+/beads for acquisition (barcoded samples = large number of cells), I am considering the possibility of omitting the final wash step in CAS+ and instead washing only in CSB (cell staining buffer) prior to acquisition.
Before running some tests to potentially implement this modification, I wanted to inquire if any of you have already attempted this or if anyone has insights into the potential consequences that removing the last wash step in CAS+ might have on the quality of acquisition.

I would greatly appreciate any advice or experiences you can share on this matter.
Thank you,
Pauline

[mleipold]

Hi Pauline,

One thing to keep in mind: the final running buffer washes (whether MilliQ, CAS, or CAS+) are there to wash away residual staining buffer salts (from CyFACS or PBS or CSB). If you don't do actually wash steps with it, there will be remaining salt when tou resuspend and dilute your sample to run on the CyTOF.

In the early days of CyTOF, we didn't do that, and would have white buildup happening on the nebulizer, injector, and cones. This was the nonvolatile PBS salts remaining. This can cause Current drift even over one long day.

And in more extreme cases, can also start to clog your nebulizer and/ injector, impacting your sample introduction efficiency (# events) and/or deflecting their path through the plasma so that they don't hit the cone orifice properly (affecting your signal intensity).

I've seen all of that happen.


Mike