Yes, you can isolate around ~0.5 to 1x10^6 cells per mouse from the peritoneal cavity without any elicitation. However the macrophages are not pure as you will have some other (B cells) populations. You can get rid of the non adherent cells by incubating 4-6 hrs.

best

You can use some other agents such as proteose peptone in place of thioglycolate. If you required macrophages without activated one then you can simply isolate it which will be small amount, but you can increase their yield by modifying protocol or optimizing it such as: collect as much peritoneal fluid as much possible in chilled tube and keep it on ice during collection. You can also use chilled PBS to inject into peritoneal cavity which will increase numbers of macrophages in peritoneal cavity. Inject 5 ml of ice cold PBS (with 3% FCS) into the peritoneal cavity using a 27g needle. (10 times more macrophages can be collected )

Hope this will help you to increase your macrophages yield into isolated sample without compromising with your experiment.

Hi Nasim,

naive peritoneal macrophages are our standard ex vivo macrophage model in the lab and we use them nearly every day.

We puplished a little paper recently that describes transfection of primary macrophages but also describes very detailed the preparation peritoneal macrophages without any pre-stimulation like bio gel or thioglycollate.

If you like, please have a look at:

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA, November 2019, Journal of Visualized Experiments,

DOI: 10.3791/60143

Please feel free to ask any further questions.

All the best and a happy new year.

Marc

thank you for all the answers.

Hi marc,

how many macrophages can we isolate from this protocol?

You can get 10

Times more cells after injecting chilled PBS i mentioned in my reply, macropahge no can be arround 7-10 million cells.

Hey Nasim,

it varies in dependency of age and gender of the mice and we also saw variations between different WT and/or knock-out lines, but in general you can calculate with 500.000 macrophages (young female) up to 1.000.000 macrophages (old male). We always use ice-cold PBS and keep all our solutions during the process on ice because macrohages start "chewing on everything" when kept on room temperature.

We did not observe that the temperature of the lavage solution had any effect on cell numbers. Before and after sorting we always make and FACS analysis (gated on CD11b-F4/80 douple positive) to prove purity and enrichtment efficancy. How many macrophages you have right after the lavage also greatly varies between mouse lines, gender and age (40-60% CD11b-F4/80). But these details and the FACS analysis are also in the paper.

I hope that further helps you.

All the best,

Marc

Thank you very much Marc