Have a look at this recent paper:

Host response. Inflammation-induced disruption of SCS macrophages impairs B cell responses to secondary infection. Gaya M, Castello A, Montaner B, Rogers N, Reis e Sousa C, Bruckbauer A, Batista FD. Science. 2015 Feb 6;347(6222):667-72.

You can see that in untreated mice there are almost no detectable macrophages by FACS. You will need to pool a lot of LN.

Smashing LN between slides is OK if you are looking for the many T and B-cells inside but NOT a good idea if you want Macs and DCs (big fragile cells). Also, macrophages are sticky cells. Try digestion in a tube and using fine scisors to cut LN open to allow digestion. Put everything on a cell strainer and gently push though with seringe rubber. 5 min centrifugation at 1200rpm should be enough.

Good luck

Thanks very much for that info, Dr. Brandt.

For the experiment I will be challenging the LN with bacteria. Not sure to what extent that will affect my macrophage numbers, but I plan to pool 4 lymph nodes for sorting.

The unchallenged lymph nodes are so small that I'm finding it really difficult to "tease them apart" before digesting, as some protocols suggest. I'll have to find something appropriate to cut the lymph nodes with.

Accurate macrophage quantification in any tissue using flow cytometry is very unreliable. We have used 2P to quantify leukocytes in the skin (http://www.ncbi.nlm.nih.gov/pubmed/25007044), and the numbers of macs actually present vs numbers released are so different that I've given up any hope that it can be done. Put simply, the migratory, non-adherent cells such as T cells, B cells and DC pretty much "fall" out of the tissue, while the stationary/adherent cells (macrophages, mast cells) remain bound up with the stroma. And matters only get worse during infection. A large phagocyte laden with bacteria will never survive the processing. Again, using 2P, we can see APC absolutely bloated with pathogens (http://www.ncbi.nlm.nih.gov/pubmed/19043558), and they just tear up whenever we try to isolate them.

You may want to consider alternative approaches. Histology is time-consuming and problematic, but it often does a better job of quantifying what's actually going on in a tissue than FACS.

That being said, if you're seeing large enough changes, I think it is fair to say that you can expect to see a difference using a FACS-based assay. And you can probably reduce your cell loss quite a bit with some minor changes to the protocol.

I would avoid using slides altogether, a lot of potential for loss. Put the LN directly into a tube with 500ul PBS and cut in half using fine scissors before adding enzymes. Collagenase IV is pretty strong, which I think you need for macs, but if you're having viability issues you can move to Collagenase D or Liberase (they're expensive though). You can also "scale-up" to trypsin if you really want to release as much as possible (note, though, that a lot of cell surface antigens are cleaved by trypsin). Also note that, unlike trypsin, collagenase is not affected by the presence of protein, so you can put the digestion mix in FBS-containing media to maintain viability. We typically digest LNs in 1mg/ml collagenase at RT, not at 37oC. We digest skin at 37oC, but the skin has a lot more collagen. Inactivate the collagenase using EDTA, and keep 5mM EDTA in your FACS buffer, it will help keep the cells disassociated from one another. Wash FACS buffer through the sieve before and after you've pushed the cells through, to prevent loss of cells on the sieve itself. Your spinning parameters are okay, though I personally use something a bit faster (1500 RPM, approx. 500g I think).

Popliteal LNs are small, and you'll be lucky to extract any more than a million cells total from a single LN of an untreated mouse.