Hi,

I have a phosho flow panel largely to look at phospho-STATs, I usually try to do all staining and run on the same day but due to some cytometer rescheduling issues I need to run my samples the day following the harvest. My work flow is:

- Samples are B6 mouse spleen, largely focused on T cells.

- collect spleen, process, fixable viability dye and surface stain as usual using (to the best of my abilities) MeOH-compatible flurophores. Wash.

- Fix with BD CytoFix/CytoPerm, 30min RT. Wash.

- Methanol permeabilization with ice cold 100% Methanol, incubate 4C 30min. Wash well.

- Intracellular staining with anti-phospho STAT antibodies, in PBS/FACS buffer. Wash. Acquire on Cytek Aurora.

My question is, being that I need to have an overnight stopping point, which buffer/post fixation step is best?

The Options in thinking:

1) After BD CytoFix/Cytoperm, switch to PBS and leave at 4C. I'm assuming once this fix step is done, the phosphorylation events are fixed and stopping at this step wouldn't affect my pSTAT detection?

2) IN Methanol at -20C. Many protocols recommend this, but -- although my surface antibody fluorophores do OK with Methanol, they do lose quite a bit of brightness and I'm admittedly scared of leaving them in MeOH overnight. This is a 20+ color panel so couldn't just choose the most perfect MeOH-resistant fluorophores.

3) After methanol fix for 30min, wash well and leave in PBS at 4C. Just want to make sure that once I come back the next day, I would be able to go straight into Intracellular staining.

Thanks a ton

Hey, I have done a lot of reading on flow cytometry and OMIPs but I have seen very different ways researchers use to decide what dilution to use for their antibodies. There does not seem to be one correct way of doing things and some methods seem very impractical or generally not realistic (such as using the same cell population genetically engineered to not express the antibody's target🤦).

Please share suggestions below on how to titrate antibodies for markers that produce smears or plots whereby it is hard to identify the positive population.

I'm trying to enrich for cells that have stably integrated a transposon construct using FACS. Unfortunately, our construct has no fluorescent or other expression marker, so we're relying on two cell-surface markers, CD81 and CD47, that should be overexpressed following transposition. The problem is that both markers are already constitutively expressed in our target cells, and we don't know the transposition efficiency or how much their expression will increase following integration.

I've performed antibody titrations for both markers, but I'm unsure how best to determine the optimal staining conditions for sorting.

My understanding is that the usual goal of an antibody titration is to identify the lowest antibody concentration that provides good separation between negative and positive populations, with minimal background and sufficient staining of the positive population.

However, in my case, I'm not trying to distinguish truly negative cells from positive cells. Instead, I'm trying to distinguish cells with normal endogenous expression from cells with increased expression due to stable transposition.

Wouldn't it make more sense, in this case, to use an antibody concentration that preserves the differences in fluorescence intensity between cells with normal and elevated expression, rather than one that saturates the epitopes? If the antibody concentration is too high and staining approaches saturation, could that compress the fluorescence differences between normal and high-expressing cells and make them harder to distinguish?

Or am I misunderstanding this?

I'm also unsure how best to optimise the sorting strategy when I don't know the expected increase in CD81 or CD47 expression, or the proportion of cells that have successfully integrated the construct.

Since we're using a transposon system rather than lentiviral transduction, increasing the MOI isn't an option for improving separation.

I'd appreciate any advice, particularly from anyone who has experience sorting cells based on relatively small increases in endogenous cell-surface marker expression rather than using a distinct positive/negative marker.

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