We spend a lot of time culturing primary macrophages, I have tried most of the different markers suggested above. As noted by several authors, CD14 is standard but you should be aware that sometimes - depending on the donor or on the culture system, you do get a loss of CD14, it is usually still around but with decreased expression - this actually happens in monocytes before they fully mature into macrophages (see Buckner et. al., 2011). CD68 is a good marker, but it being intracellular makes it a little trickier to stain for by FACS. I would avoid CD163, as this one can change with activation (and activation can change with culture conditions), and we have used the transferrin receptor (CD71) but had mixed results.

In terms of using flow, if you have to pick up your macrophages, you are going to get 20 - 40% cell death and a lot of debris, but you can certainly do it. I have not had much luck with Accutase, but have been able to use the reagent TrypLE Express (from Invitrogen) which works pretty well. I have also used Versene in the past, but that is very harsh. And I know a lab which uses Lidocaine and swears by it.

One thing to consider here is the length of time you let the MDM adhere. If you are using the adherence just for purification, you can probably do it for a shorter time (between 1 and 4 hours) and may have an easier time picking the cells up ... but if that is the case, you might try using magnetic beads to separate out the cells. Adhering the cells for longer (days) will generate a very pure population, as everything else will wash away, but after several days in culture MDM are very hard to lift.

CD-14 beads!

Hey Ashley, how would you like to check the purity of your monocyte-derived macrophages? By flow cytometry?

By Flow Cytometry you can use the same markers! But you will have to trypsinze the cells.

yes and here the problems can start. you shouldn't trypsinize macrophages (https://www.researchgate.net/post/What_is_the_best_way_to_harvest_adherent_macrophages_out_of_48-well_plates)

You may also proof the purity of macrophages by immunofluorescence of adherent macrophages. but by this method you will hardly be able to use immunofluorescence stainings against surface proteins (like CD14) as a marcer.

But i now saw, one of Ashley's Topics is "Flow Cytometry". So i also would recommend CD14 for Flow Cytometry.

By gene expression u should have high expression of CD163, LXRalpha, APOE and FCgammaR1 and low of WNT5A and SOCS1.... This is what we are currently doing now and once we know that they are macrophages then we wil go for immunostaining using antibodies

There are good antibodies for CD14, CD163, CD68, CD71. I think the "best" marker will depend upon how they differentiated. Primary alveolar macrophages CD14 or CD163. However, after stimulation with phorbol esters, CD163 is shed. For mdm, one of the other markers likely better (CD68, CD71). FCgamma receptors may also be useful (CD16, CD64)

Thanks everyone! I will look into all of your suggestions. I would like to use flow, but I've been told that most of the cells die when you lift them from the plate, so I will also have to look into alternatives to trypsin as well.

We spend a lot of time culturing primary macrophages, I have tried most of the different markers suggested above. As noted by several authors, CD14 is standard but you should be aware that sometimes - depending on the donor or on the culture system, you do get a loss of CD14, it is usually still around but with decreased expression - this actually happens in monocytes before they fully mature into macrophages (see Buckner et. al., 2011). CD68 is a good marker, but it being intracellular makes it a little trickier to stain for by FACS. I would avoid CD163, as this one can change with activation (and activation can change with culture conditions), and we have used the transferrin receptor (CD71) but had mixed results.

In terms of using flow, if you have to pick up your macrophages, you are going to get 20 - 40% cell death and a lot of debris, but you can certainly do it. I have not had much luck with Accutase, but have been able to use the reagent TrypLE Express (from Invitrogen) which works pretty well. I have also used Versene in the past, but that is very harsh. And I know a lab which uses Lidocaine and swears by it.

One thing to consider here is the length of time you let the MDM adhere. If you are using the adherence just for purification, you can probably do it for a shorter time (between 1 and 4 hours) and may have an easier time picking the cells up ... but if that is the case, you might try using magnetic beads to separate out the cells. Adhering the cells for longer (days) will generate a very pure population, as everything else will wash away, but after several days in culture MDM are very hard to lift.