u/Sufficient-Cry-541

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

I love this table from Thermo but unfortunately they don't list or have tested mouse IL-6 or GM-CSF staining. Anyone know if the Foxp3 kit is compatible with staining mouse IL-6 and GM-CSF?

For details general work flow is: in vivo mouse model that's very inflammatory, pulling out spleen and processing to single cell suspension, will be doing 4hr PMA Iono stim, would ideally like to use the Foxp3 kit bc I have a couple of intranuclear targets and would ideally not have to split into a separate panel using CytoFix/Perm. But if anyone has experience with staining for either of the targets above with the nuclear kit, would love to hear how it went.

Thanks!

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/cell-analysis-resource-library/ebioscience-resources/intracellular-staining-buffer-selection-guide.html