Probably the most frequent question I get about overnight staining is along the lines of "won't my cells die?" So today, I'm going to discuss viability in overnight staining.
Fixable viability dye ViaKrome 808 staining on mouse blood.
Before I get into the details, I want to clarify two things:
1) The vast majority of the overnight staining I do is on fixed cells. Viability is not an issue there because the cells are already dead.
2) For unfixed cells, you want to keep the cells cold (4C) and handle them gently.
At the end of this post, I'll make some general recommendations for best approaches depending on the species you're working with and which kinds of markers you're staining for.
Viability is really critical to getting good data in Flow Cytometry regardless of the approach you're using. Although we can exclude dead and dying cells with viability stains, if the prep has low viability, the results are often weird and there can be more non-specific binding. Some cells tend to die more easily than others (looking at you, neutrophils and Tregs), so in addition to staining artefacts, there can be changes in the distribution of cell types. This is really a problem if you're dealing with cells from different sources, say tumor versus peripheral blood, and the isolation methods lead to some cell death.
Let's have a look at samples with high and low viability to see how the cells survive overnight.
In this example, I've prepared cells from C57BL/6 spleen and the small intestinal lamina
propria (the gut). The gut prep is deliberately suboptimal. The methodology here is a 20min viability stain with one fixable viability dye (eFluor780), an overnight incubation at 4C (in the fridge, in the dark), then a second viability labelling with a different dye (ViaKrome 808).
Here's the starting viability for CD45+ leukocytes in both preps (n=3 mice):
I've then compared how the cells survive in various buffers. PBS with 0.5% BSA and PBS with 2.5% fetal calf serum are two fairly common definitions for so-called FACS buffer. I always include 2mM EDTA in mine to prevent cell clumping. After that, we've got HBSS, DMEM, RPMI and IMDM as a base with 2.5% FCS added. These all contain things like glucose to keep cells happy. These media are all buffered with HEPES since they won't be in a CO2 atmosphere. After the staining, the cells are all washed with PBS prior to staining for the second viability stain.
In the graph below, the viability is assessed as a fraction of the cells that were negative for the first viability dye. What that means is, we're looking at how many cells survived the
overnight staining, not counting those that were dead to start with.
The spleen viabilities stay high, while the gut samples that were already dying tend to keep dying. This is for total CD45+ leukocytes. What about specific cell types?
Well, here's a breakdown for major cell types from the spleen:
Notably, Tregs suffer badly in PBS, and HBSS is pretty terrible. Surprisingly, neutrophils did just fine.
If you're going to use unfixed, fresh cells for overnight staining, I recommend using DMEM or IMDM with 2.5% FCS, buffered with HEPES. You can add 2mM EDTA to prevent clumping and up to 0.1% sodium azide to stop metabolism. As long as the cells are kept cold, there should be no metabolic activity or change in protein expression.
As I said at the beginning, however, most of the time I use fixed cells. There are two reasons for this: 1) I frequently work with cells extracted from tissues like the gut where the viability isn't amazing to start with, and 2) I almost always want to stain for surface and intracellular antigens, so by fixing, I can do both in one step.
Here are my recommendations for some situations. If the situation you're interested in isn't covered, ask and I might have an answer.
Human cells, surface epitopes only: Stain 20min for viability dye in PBS or FACS buffer, fix 20min with 0.2% formaldehyde (formalin, not PFA), stain overnight in the buffer of your choice.
Mouse cells, surface epitopes only: Stain 20min for viability dye in PBS or FACS buffer, then stain overnight in DMEM with FCS, etc. This is the best option for many chemokine receptors.
Human cells, mixed surface and intracellular targets: Stain 20min for viability dye in PBS or FACS buffer, fix 20min with 0.2% formaldehyde (formalin, not PFA), stain overnight in the buffer of your choice. Then, wash, fix again with the eBioscience™ Foxp3 / Transcription Factor Fixation/Permeabilization buffer, stain overnight in the permeabilization buffer from that kit for intracellular epitopes.
Mouse cells, mixed surface and intracellular targets: Surface stain for 1hr for viability and any epitopes that are ruined by fixation (e.g., CD69, CCR9), wash, fix with eBioscience™ Foxp3 / Transcription Factor Fixation/Permeabilization buffer, stain overnight in the permeabilization buffer from that kit.
Cytokine (plus nuclear) staining: There are additional details here that I'll cover in a separate post, but the best protocol is as follows: Surface stain for 1hr for viability and any epitopes that are ruined by fixation, wash, fix with 2% formaldehyde (not PFA) for 40min at room temperature in the dark, stain overnight in the permeabilization buffer from that Foxp3 kit. Note: many more epitopes are affected by formaldehyde than by the eBioscience fix/perm kit.
Phospho-proteins (plus nuclear): Again, there are additional details here that I'll cover separately with the protocol. This one is really different from the others due to the stimulation and permeabilization requirements. Stain for viability before stimulating your cells. Surface stain with synthetic dyes only in the 5-30min stimulation at 37C. Fix in the stimulation medium by adding 4% formaldehyde (neat formalin) to reach 2% final concentration. Wash, permeabilize with -20C methanol. Wash with TBS-FCS-azide (not PBS-based buffers). Stain overnight in TBS-based buffer at room temperature in the dark.
Fluorescent proteins, surface antigens only: Again, there's are some important details I'll address later. For this situation, your best bet is generally to stick to shorter staining times. This can be done with the overnight surface staining.
Fluorescent proteins plus intracellular epitopes: Surface stain for 1-4hr for viability and any epitopes that are ruined by fixation, wash, fix with 2% formaldehyde (not PFA) for 40min at room temperature in the dark, stain overnight in the permeabilization buffer from that Foxp3 kit. Formaldehyde is important for retaining the GFP/YFP/RFP/mCherry signals, but wrecks a lot of epitopes, so you'll probably want to stain for most of your surface antigens before fixation.
What about cryopreserved PBMCs? The viability of PBMCs after thawing can vary tremendously depending on how they've been treated. I've done quite a bit over overnight surface staining on unfixed frozen PBMCs with good results, but in general, the safer option is to use the quick, mild fix with 0.2% formalin.
I hope that helps you get started. In the next post, I hope to cover details about specificity and how to go about re-titrating your antibodies for use in overnight staining.
Reagents mentioned:
While I'm now embedded in the Department of Pathology at the University of Cambridge, most of the work I'll show was done at the Babraham Institute in Cambridge, the University of South Australia in Adelaide, and at the VIB/KUL in Leuven, Belgium. I'd like to thank the flow cores and staff at those institutions for their help and support. The research that led to the development of this method was made possible by the ERC and the Wellcome Trust.
Hey Oliver,
thanks so much for your posts. How you prepare 2%FA vs 4%PFA? Thanks
Your protocol for O/N staining is terrific and I am starting to implement it. Regarding the Fc blocking step, some people perform it 10min then wash and add the antibodies, that is what I usually do, and others perform it at the same time of the staining. How do you deal with it in your O/N protocol?
I love what you're doing here. I notice you only bring up fixation ruining epitopes at higher concentrations of formalin, is this not a concern with 0.2%? 0.2% seems to be a magical light touch that preserves the cells overnight without affecting antibody binding?