a) Thio: as people suggested - age your thio for at least one month (http://www.ncbi.nlm.nih.gov/pubmed/9050940). PBS vs water - does not matter, we've got slightly better yield using thio from BD (in comparison to Sigma). Also: do not use young mice (younger than 2 mo), older/bigger mice give higher yield.

b) Harvesting time: 3-5 days gives the highest number of cells, % of macrophages is slightly higher at day 5.

c) F4/80 is not specific for macs, it is also expressed on eosinophils. Three-five days after thio ~35-40% of all cells in peritoneal cavity are eosinophils (http://www.ncbi.nlm.nih.gov/pubmed/22706315). Even after enrichment by adhesion to the culture dish some non-mac cells can be retained. Depending on how much purity your need for your expt you may want to deplete your thio-elicited cells using MACS or sort macrophages by FACS.

Did you directly do FACS analysis of peritoneal exudates or did you culture it overnight for adherence. In my experience after culturing overnight, you get 90% macrophages. You still get F4/80 positive and negative cell, however could change after removing all the non-adherent cells. Hope this will help

In our experience we have found on day four to have a population of 80% F4/80+ macrophages with 15% F4/80+ eosinophils. Do you mature your thioglycollate?

I do filter sterilise the thioglycollate (but not autoclave it) and let it stand for more than a week. Does the autoclaving make the big difference?

I checked some protocols online. They prepare their 3% thioglycollate in distilled water (whereas I prepared mine in PBS). Would this be why I'm not getting macrophages?

I prepare 3% thioglycollate in d/w and autoclave for 15mins. You get more macrophages if you use thioglycollate of old batches than recently prepared. Normally, you should collect peritoneal macropahges within 3-5 days after thioglycollate inoculation. The best we get is on 5th day. I get more than 90% of macrophages. good luck

One potential problem is that some thioglycollates are now vegetable based not bovine broth based. We found significant differences between the two. One should only use the bovine broth based thioglycollate. Another possibility is that you truly have 2 populations of macrophages. Not all macrophages will be F4/80+ regardless of the tissue source.

Mature your thioglycollate and it will help. Also not sure why using Ly6G( is this a marker of neutrophils?). should be Ly6C instead if you are looking macrophage, or using CD11b.

I used Ly6G as a neutrophil marker to check i got no contamination

I agree with Dominic. We routinely get 3 x 10^6 resident cells about 90% macrophages (by microscopic examination of cytospin preps stained with Wright Giemsa). I also agree that thioglycollate needs to be prepared with dH2O, not PBS. It is designed as a bacterial growth broth, so already has salts in it, I think.

One more thing. In doing peritoneal lavage, we use cold PBS with 1% fetal bovine serum. Both the low temperature and the presence of protein seems important in harvesting the cells

There will usually also be lymphocytes and mast cells in the peritoneal wash. You might want to do a Wright stain on the cells you get to see what they look like. Since thioglycollate varies from different manufacturers that may be affecting your result. Try sampling at times after injection, starting at 72 hours to see if you can maximize your return of macrophages.

For peritoneal lavage I inject/extract 5mL of cold Hanks + 2%FBS a total of 3 times in to untreated mice and routinely get 3-4X10^6 cells with about 90% of them being macrophages for tissue culture.

you can also try using 5% starch solution in physiological saline instead of using thioglycolate. try doing the lavage with ice-cold RPMI culture media instead of PBS. If it is not affecting to experimental protocol, you could also try to harvest the cells between 72h-96h post thio/starch injection to get a high yield (thats what i get from BALB/c). the exudate cells that you get are of a mixed population (monocyte/macrophages/lymphocytes). however, i usually find approx 70% CD11b positive cells from the total exudate. if you need only macrophages, incubate cells in tissue culture petri dishes for 6-8 hrs in a 5% CO2 incubator at 37 degrees. all the monocyte/macrophages will adhere to the plate whereas other cells will remain floating which can be discarded by washing. you can harvest the adherent cells by treating with trypsin or accuatse.

The two distinct macrophage populations you see resemble the populations described by Ghosn et al PNAS 2010. After thioglycolate injection, small peritoneal macrophages that express high levels of MHC class II but very low levels of F4/80 predominate.

10-12 ml ice cold media (HANKS or PBS) improve cell harvesting

and

With 4-5 hr culture of harvested cells, macrophages will adhered to the tissue culture plate and other cells could be washed, the remaining cells are more than 90% macrophage

Thanks for all the suggestions everyone. Appreciate it!

You collect PEM too late. Based on my experience, the optimal time is the 4th day after Thioglycollate injection. At 6th day the inflammation is already largely resolved. The double negative population the most likely represents lymphocytes.

If six days post thioglycollate is a experimental condition, you could try Percoll discontinous gradient to enrich macrophage subpopulation based on F4/80 positivity. Be sure that these two distincts populations are also different regarding functional abiliites.

a) Thio: as people suggested - age your thio for at least one month (http://www.ncbi.nlm.nih.gov/pubmed/9050940). PBS vs water - does not matter, we've got slightly better yield using thio from BD (in comparison to Sigma). Also: do not use young mice (younger than 2 mo), older/bigger mice give higher yield.

b) Harvesting time: 3-5 days gives the highest number of cells, % of macrophages is slightly higher at day 5.

c) F4/80 is not specific for macs, it is also expressed on eosinophils. Three-five days after thio ~35-40% of all cells in peritoneal cavity are eosinophils (http://www.ncbi.nlm.nih.gov/pubmed/22706315). Even after enrichment by adhesion to the culture dish some non-mac cells can be retained. Depending on how much purity your need for your expt you may want to deplete your thio-elicited cells using MACS or sort macrophages by FACS.

Can you attach an image of your flow charts, including fsc/ssc, f480 vs ssc&fsc, and f480 vs. ly6g? I agree that day 4 is ideal. I have noticed a negative population that is similar in size to lymphocytes, but negative for lymphocyte markers, as well as ly6g & f480.