For the specific purpose you mention, I would strongly advise against freezing your tissue for later processing. Two main issues come to mind:

1) Quality of flow cytometry data strongly depends on cell viability. Not all cells survive cryopreservation, and different cryopreservation media may adversely affect specific cell populations or markers.

2) Frozen cells may not behave as expected after thawing. This is a critical point if you are planning to characterize macrophage populations in the kidney - they may produce more cytokines than fresh cells, which would be an artifact of using frozen cells and could lead to scientific misinterpretation. .

However, I do empathize with your problem (been there!). If using fresh cells is totally out of the question, your best option is to run pilot experiments to assess the impact of different preservation methods in the FACS results you get from fresh versus frozen cells.

Alternatively, you might want to try keeping the kidneys in the fridge overnight for processing next day; check this conversation in Researchgate:

https://www.researchgate.net/post/Can_mouse_tissues_used_for_immune_cell_isolation_be_stored_over_night

I tried it myself with lungs and spleens and, although I did got less yield, the balance between the main immune cell populations seemed unchanged. However, I wasn't looking at activation markers, so I would proceed with caution if you want to know the activation status of kidney macrophages.

As previously suggested, freezing whole organs is not recommended esp. when doing flow cytometry. Even if you use specialized freezing solutions, these are usually meant for downstream RNA/DNA analyses and are not suitable for flow cytometry. Below are your 3 options in my order of preference.

1. You could process the kidneys, perform your staining, fix your cells and then run flow the next day.

2. Place the whole organ in complete medium and leave overnight in the fridge. The next day, process, stain and fix. You could do flow the same day or the next. You may have perturbations in immune cell proportions and function but these should not be a big deal if you minimize the amount of time between collection and processing (collect late in the day and then process early the next day) and if your organs are intact.

3. Process the kidneys, isolate cells and then freeze them. You can stain and analyze the cells later. The problem here is that you may lost sensitive cell subsets during freeze-thaw and immune cell functionality goes down with freezing.

Thank you very much. I will try the methods mentioned and see which one gives the best results.