How did you get the mice death? This is an important point for the following experiment. The best methods are: Carbon dioxide gas inhalation and anesthetic

injection overdose. An other point, you do first injection with 0.5 mL with ice-cold Mg- and Ca-free PBS-EDTA (0.6 mM), then aspirate and eject the saline solution in a falcon 50 mL tube. Then a minimum of 8 washes is required for recover 0.2-05 x10^6 cells per mouse with quite good AM purity and 96% viability.

If you are working with mice you can normally expect 300 000 AM from one mouse with PBS lavage.

You can try to sort CD45+/CD11c+/CD11b-/Siglec-F+/Gr-1- cells or alternatively pool mice... we did both a few times and it worked great.

Hi. How much macrophages do you get from one mouse? How do you know the alveolar macrophages adhere to the epithelium?

Use ca mg free pbs with 2 mM EDTA to get higher yields.

The lavage buffer must not have divalent cations in it, that reduces yield.

Some report better yields with 0.6 mM EDTA to further remove cations.

Typical yields for mice in a barrier facility are indeed in the 200-300K range.

Pooling is probably the best approach. The yields from large mammals (pigs and cattle) are large-as the lavage volume is greater. Will work better if the lavage buffer is warm.

Agree with Gyorgy Fejer. Can use rat to get more yield.

Thanks very much, I will pass this on to my colleague! He has only been getting around 70,000 cells from a mouse so there must be a problem somewhere! I will suggest adding EDTA and using no PBS with no calcium/magnesium. Thanks again!

I typically see 2-3E5 alveolar macs from one mouse. I usually pool cells from multiple mice to get enough for an experiment. I have had luck with using 0.5mM EDTA/PBS for the lavage. And I try to lavage 3-4 times per mouse to get the macs out.

Hi,

I asked our asthma mouse researchers who do this several hundred times a week. They use 3 x 400µl PBS/2mM EDTA to flush mouse lungs. However, in non- inflamed lungs, like Gyorgy pointed out, you cannot expected more then 200.000-300.000 total cells (most of them AM of cause).

We use 1 ml of ice cold PBS, gently massage the lungs, withdraw the lavage fluid and repeat 3-4 times. We recover approx. 1 million/mouse using this method. If you just want to use BAL without massage, one will get about 80,000-160,000 cells using multiple washes. You can also try HBSS to make the cells a little happier.

Alveolar macrophages and peritoneal macrophages belong to resident macrophages. In order to get high yields, the following two points are critical. Firstly, the lavage buffer would be at 4℃ before use. Secondly, the lavage buffer is Ca2+ and Mg2+ free. We used ice cold Ca2+ and Mg2+ free PBS with 0.2%EDTA.Na2 to wash perittoneal cavity(3-5ml, three time) for harvasting peritoneal macrophages. Pooling is another optional.

From bronchoalveolar cavity 70,000 cells is a good number for mouse.

From peritoneal cavity, you can have around 300,000 macrophages.

Will work better with cold PBS

How did you get the mice death? This is an important point for the following experiment. The best methods are: Carbon dioxide gas inhalation and anesthetic

injection overdose. An other point, you do first injection with 0.5 mL with ice-cold Mg- and Ca-free PBS-EDTA (0.6 mM), then aspirate and eject the saline solution in a falcon 50 mL tube. Then a minimum of 8 washes is required for recover 0.2-05 x10^6 cells per mouse with quite good AM purity and 96% viability.

I usually proceed with Pento-barbital OD, then washed with Ice cold PBS (Mg 2+/Ca2+ Free) and after I wash gently the lungs several time. I let the lung resting when full of media to increase the cell recovery. I usually get around 2.5.10^5 cells.

As described in my paper ,

lavage buffer you could use Ca2+ and Mg2+ free. ice cold PBS with 0.2%EDTA.Na2 For harvesting peritoneal macrophages. Use syringe needle appropriate . Rub the lungs gently during harvesting and make sure body is still warm. Decapitation of the mice can be used and immediately be ready to inject PBS for harvesting . Pooling is another critical and should be kept on ice cold tube and process right away for culturing .We used Leighton tube for culturing. Hope it helps

Have you thought about digesting the entire lung in collagenase D and using plastic adherence or MACs sorting afterwards?

I agree with Carol Gardner

I have used Ca and Mg free HBBS (as Seppe Vander Beken) supplemented with 0.1 mM EDTA at RT to Isolate alveolar macrophages from adult pig lungs. Filled the lungs (several litres!), palpated aggressively for about 10 minutes and decanted. Gives about 10^8 macrophages which we used in immobilization tests. Not sure how this would translate to the mouse.

My previous experience with resident peritoneal MΦs suggests that ice cold PBS/EDTA 2mM is sufficient to generate a good yield of these cells. For alveolar MΦs I ve used saline before.

Low yield doesn't necessarily mean that they are adhering. Make sure your suture is tight around the endotracheal tube, push 1mL volume in slowly, and extract even slower. Put first mL into collection tube and get fresh fluid for 2nd and 3rd extractions (slightly less than 3mL total volume when finished). The idea here is not to ruin the architecture of the lung by forcing fluid in and out. Think of it as a series of valves that can be broken very easily. Gently push them in and gently pull them out. I have always used blood bank buffered saline (BBBS) for extraction and isoflurane for overdose. Roughly, 200,000 cells per mouse with high viability.

Try using Cal Mag free PBS. The presence of Cal and Mag encourages the rentention and expression of cell surface proteins. Once the cells are collected you can replace with Media etc....

A lot of people mentioned it before: ice, ice, ice, cold tubes etc. Very important! Also EDTA and other substances to prevent attaching or to detach the AM...

Can't we get human macrophages from Bronchoalveolar lavage and culture them?

sorry for my naiveté….

Any questions regarding macophage?pl shhot me email or write

Ice definitely, but for our removal from the animal, we use Heparin 10U/mL. If you rinse the cells a couple times with a Balanced Phosphate/ salt buffer (BSS) the resulting activated macrophages adhere very well. I'm not sure if Heparin will be a problem for your cells for future use, but just to get them off the animal tissue it works very well.

Thank all of you for your kindly answers!  The methods  benefited me a great deal.

Try 3-4 rounds of washes with 700 ul PBS/10 mM EDTA. There s a paper coming out very soon where we have used this protocol: Kapellos et al. (2016) Biochemical Pharmacology.