My protocol is as follows:

Seed 5 x 10^6 bone marrow cells in a non tissue culture treated but sterile 10cm petri dish on day 1 with DMEM, high glucose, GlutaMAX supplemented with 10% FBS, 2.5 mM β-Mercaptoethanol, 100 U ml-1 penicillin and 100 mg ml-1 streptomycin supplemented with 1% additional non essential amino acids (we buy in 100x solution from Gibco), and 20% L929-cell conditioned medium. We don't actually use M-CSF in our lab we use L929 conditioned media. L929 cells are a fibroblast cell line that produce M-CSF. They also produce a lot of other stuff that seem to help them differentiate. They are also quite metabolically active as well so make sure they have enough amino acids and glutamine. I found out by accident the other week by picking up the glutamine negative media then trying to supplement it a few hours later when I realised all the cells died. So primary mouse cultures are pretty picky about their media. 

On day 2 I add 10ml more of media to the dish so the total volume is 20ml. Do this very gently so you don't disturb the bone marrow cells that are lightly stuck down to the dish. 

On day 4 remove 10ml of media, spin the media down, there shouldn't be many, if any,  cells in the media by this point they should all have become adherent to the plate. If not you can re-suspend in 10ml of fresh media and add back to the culture. Do NOT remove all the the media. The differentiating cells are producing a lot of autocrine signalling compounds, if you remove it all you remove all of these and you will disturb the differentiation.  Do this whole process as gently as you can. I've found through experience the less you touch and disturb the cells the better they differentiate.  Don't pick them up to look at them every day between changing the media just leave them alone.

By day 6 you now have fully differentiated cells which can then be polarized using a stimulant.  I normally have bout 80% confluent at this point. It sounds as if  you are not seeding enough cells in your plate. 

As they are differentiating the cells do not proliferate very well. You should end up with less cells than you have seeded as not all will differentiate into macrophages.  As macrophages emerge in the culture they will eat any other cell types that are around and any cells that have not made it though the differentiation process. Naturally you wont get 100% differentiation. I tend to get between 85-90% differentiation. Compared to RAW cells they are very sluggish and slow and will appear different in morphology. From my experience RAW cells tend to look very triangular sometimes and they are quite homogenious in culture, BMDM are not. They are very hetrogenious and quite round and flat until you stimulate them, then they go crazy.

You can polarise them using a number of stimuli. I tend to study signalling so I do quite weak short term stimulations. I tend to go for 100ng/ml of LPS, though you can also use interferon gamma and LPS together to really give them a boot so to speak. If you are looking for the "classical" M1 activation phenotype these are the two to go for. I've also done a lot of IL-4 and IL-10 stimulation on BMDM for M2 polarisation and I tend to use around 20ng/ml or 10ng/ml dependent on the situation.  If you're looking at signalling anything from 1 to 30 minutes up to an hour if you're looking at cytokine production etc more like 6 hours or so up to 24 hours. 

The cells are plastic in terms of response up until you differentiate them. Differentiated cells on day 6 but with no stimuli don't really do anything. They just sit around waiting for something to tell them what to do. 

For best results play around with your culture conditions. I don't like using recombinant MCSF I much prefer to the L929 conditioned media as I feel they produce better results, also play around with your seeding density. I found 4x10^6 was too little and my cell cultures collapsed as they need to be around other cells to survive. Try seeding a few plates between 3 - 6 x 10^6 and see if it helps increase your yield. 

I'm sorry for the huge length of the reply but I hope this helps, if you have nay more Qs feel free to message me I'll try help the best I can. 

My protocol is as follows:

Seed 5 x 10^6 bone marrow cells in a non tissue culture treated but sterile 10cm petri dish on day 1 with DMEM, high glucose, GlutaMAX supplemented with 10% FBS, 2.5 mM β-Mercaptoethanol, 100 U ml-1 penicillin and 100 mg ml-1 streptomycin supplemented with 1% additional non essential amino acids (we buy in 100x solution from Gibco), and 20% L929-cell conditioned medium. We don't actually use M-CSF in our lab we use L929 conditioned media. L929 cells are a fibroblast cell line that produce M-CSF. They also produce a lot of other stuff that seem to help them differentiate. They are also quite metabolically active as well so make sure they have enough amino acids and glutamine. I found out by accident the other week by picking up the glutamine negative media then trying to supplement it a few hours later when I realised all the cells died. So primary mouse cultures are pretty picky about their media. 

On day 2 I add 10ml more of media to the dish so the total volume is 20ml. Do this very gently so you don't disturb the bone marrow cells that are lightly stuck down to the dish. 

On day 4 remove 10ml of media, spin the media down, there shouldn't be many, if any,  cells in the media by this point they should all have become adherent to the plate. If not you can re-suspend in 10ml of fresh media and add back to the culture. Do NOT remove all the the media. The differentiating cells are producing a lot of autocrine signalling compounds, if you remove it all you remove all of these and you will disturb the differentiation.  Do this whole process as gently as you can. I've found through experience the less you touch and disturb the cells the better they differentiate.  Don't pick them up to look at them every day between changing the media just leave them alone.

By day 6 you now have fully differentiated cells which can then be polarized using a stimulant.  I normally have bout 80% confluent at this point. It sounds as if  you are not seeding enough cells in your plate. 

As they are differentiating the cells do not proliferate very well. You should end up with less cells than you have seeded as not all will differentiate into macrophages.  As macrophages emerge in the culture they will eat any other cell types that are around and any cells that have not made it though the differentiation process. Naturally you wont get 100% differentiation. I tend to get between 85-90% differentiation. Compared to RAW cells they are very sluggish and slow and will appear different in morphology. From my experience RAW cells tend to look very triangular sometimes and they are quite homogenious in culture, BMDM are not. They are very hetrogenious and quite round and flat until you stimulate them, then they go crazy.

You can polarise them using a number of stimuli. I tend to study signalling so I do quite weak short term stimulations. I tend to go for 100ng/ml of LPS, though you can also use interferon gamma and LPS together to really give them a boot so to speak. If you are looking for the "classical" M1 activation phenotype these are the two to go for. I've also done a lot of IL-4 and IL-10 stimulation on BMDM for M2 polarisation and I tend to use around 20ng/ml or 10ng/ml dependent on the situation.  If you're looking at signalling anything from 1 to 30 minutes up to an hour if you're looking at cytokine production etc more like 6 hours or so up to 24 hours. 

The cells are plastic in terms of response up until you differentiate them. Differentiated cells on day 6 but with no stimuli don't really do anything. They just sit around waiting for something to tell them what to do. 

For best results play around with your culture conditions. I don't like using recombinant MCSF I much prefer to the L929 conditioned media as I feel they produce better results, also play around with your seeding density. I found 4x10^6 was too little and my cell cultures collapsed as they need to be around other cells to survive. Try seeding a few plates between 3 - 6 x 10^6 and see if it helps increase your yield. 

I'm sorry for the huge length of the reply but I hope this helps, if you have nay more Qs feel free to message me I'll try help the best I can. 

Please don't be sorry for the lengthy reply. Detail is better then being vague! Would you be able to post a photo of differentiated, but not polarised cells? 

Hi Adrian 

http://i58.tinypic.com/33m8mfb.jpg

I've linked a photo of mine and what they look like differentiated but unpolarised, its not great as I took it down my phone on our tissue culture room microscope but you can see the cells are quite heterogeneous, some are round, some are triangle shaped. Once you stimulate them they will change morphologies and go the more classical spikey triangle or starfish looking macrophages. 

Thanks for this. I have a feeling that there is something not quite right with our culture. Our cells look much less adherent than this. In addition the confluence is much lower. I will try a higher number of cells.

Do you have any experience with freezing/thawing excess Marrow cells?

Hello

Agree with Samantha Le Sommer ,this is a good protocol you can use .

for freezing/thawing excess Marrow cells, please see links here i hope help you

Good luck

https://books.google.com.sa/books?id=P-aPIFIEM_0C&pg=PA335&lpg=PA335&dq=freezing+AND+thawing+excess++marrow+cells&source=bl&ots=OL8mX504Rb&sig=ByKGha9B25CIPkH9zr4MxDkkGvU&hl=ar&sa=X&ved=0CDQQ6AEwAmoVChMI6tPQmvnlxgIVA_EUCh1avgXl#v=onepage&q=freezing%20AND%20thawing%20excess%20%20marrow%20cells&f=false

http://onlinelibrary.wiley.com/doi/10.1111/j.1423-0410.1992.tb01213.x/abstract

https://www.researchgate.net/post/Can_you_use_cells_for_experiments_immediately_after_thawing

I routinely freeze and thaw my bone marrow, I just spin the cells down once you've finished isolating them and resuspend them at about 5 x 10^6 per ml in 90% FCS an 10% DMSO and then freeze down in a cooling box in a -80C freezer and 24 hours later transfer to cryostorage. The only thing you have to watch is when you resuscitate the cells be fast they hate the DMSO so as soon as you thaw them you have to add 20ml of room tempreture media, dribble it down the side of the falcon slowly so you don't heat shock the cells then spin immediately. 

If your cultures arn't looking quite right I'd increase your seeding density, also are you getting rid of the red blood cells in the bone marrow before you count them and put them into culture? 

dear Adrian Kaczmarek

i worked with bone marrow cells and we converted bmc cells into dendritic cells. here the problem is, bone marrow is rich in multipotent stem cells means they can convert into any type of immune cells depending upon the growth factor we are adding. here waht we are doing is giving conditions to defferentiate into desired cell type, it means the cell is undergoing differentiation...they can not proliferate ...if you need more cells you seed more number of bmc...RAW264.7 are transformed macrophage cell lines so the are immortal they can proliferate like any thing ..so we can not compare prymary cell growth rate with cell lines....thank you

Dear Sam,

Your advise was perfect. We scaled down the procedure to a 24 well plate for optimization, and found there was a strong influence on cell number. We found a lower number of cells did not support the growth of each other as well as a higher number.

In this instance we used frozen BM, thawed and used to form BMDM.

We changed the protocol that we used till now to clear the media of all the non attached, dead, useless cells. On Day 4, many cells have adhered and appear macro-phage like, but many floaters.  We collect the media the cells have been growing on, spin out the cell debris and non attached cells. During this time we gently rinse the cells in complete medium (pre-warmed, CO2 gases over night, etc), and then replaced me clarified "conditioned" media, back onto the cells, with fresh media added (50:50).

On Day 6, we had a confluent monolayer of happy healthy cells.

We will now up scale to 35mm dishes (required number of cells).

To reply to your comment Srinu, the BMDM continued to proliferate perfectly fine between day 4 and day 6. Just because they are primary cells, does not mean they stop their cell cycle.

We are running an xCelligence now to test for the proliferation rate of these cells.

Hi Adrian,

So glad I could help, there's nothing better than a nice plate of happy healthy cells! Hope your experiments go well! 

Hi Samantha, 

Do you have a good protocol or suggestions for getting the sticky buggers of the plastic. I have tried 5-10mM EDTA in PBS, but its not worked very well, even after 15-20 minutes at RT?

For RAW264.7 cells I use cell scrapers, but I'm not sure these cells like it much (tried it...).

I'd like to avoid harsh enzyme methods like trypsin/tryple to preserve surface binding proteins (doing adhesion assays with these cells).

Adrian

What ever you do do not scrape the cells! You'll kill them they really hate it. Grow them on non tissue culture treated plates, I normally use 10cm sterile bacteriological petri dishes with stem cell Accutase https://www.lifetechnologies.com/uk/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html

I normally take the cells out the incubator and sit them in the TC hood for a while before I start. It lets them cool down gently without heat shocking them. All the reagents for harvesting should be cold. If the cells have been stimulated they will be really sticky so you can also sit the petridish on ice for this should help detatch them. 

Remove the media and place it in a falcon, wash the dish ice cold PBS and put in the falcon. I keep mine in the fridge the cold will help disassociate the cells. Just make sure you wash the cells well at this stage, all the serum protein needs to be removed. You'll find that if the macrophages are unstimulated they should lift off the plate by itself. Add cold accutase to the dish, what ever you do don't warm it, it's heat sensitive and I've found that warming it up makes it less effective. Leave for about 10 minutes and you can tile the dish around and tap the bottom and they should have come off, if not leave it for longer. Dilute it with PBS and then wash the plate with PBS again, you might have to do it a couple of times but they should come off. 

You can leave the accutase on for up 20 mins and sometimes had to do the treatment twice.  I get recovery about 90% +  and the key to it is just patience, give yourself plenty of time for it because its not a quick job!  It's very gentle, from what I've seen on FACS it doesn't really affect surface protein expression. I've also done adhesion assays down stream of this protocol and they work fine. 

We are defiantly culturing on non TC plates, but they stick anyway. I agree that the scraping did not do them too well.

So cold  will detach them faster?  nice to know, and will give it a try.

I want to preserve their integrins and other attachment surface proteins so as you can imagine, gently is more important.

Unstimulated cells normally come off with just the cold PBS after a few minutes on the ice, you do have to wash quite vigorously sometimes, I use a 10ml strippette with the speed setting at max. Stimulated cells are just a pain to get off, if you find they're still sticking add some EDTA to the PBS, all three combined should get the little buggers off. 

Hi sam,

What plastic do you grow your cells on. Specifically the brand and cat number of the plates. We are finding inconsistency in growth on BD falcon plates, but great growth on corning plates.

Specifically, the macrophages seem to grow only on parts of the plate, with strong growth in groups of cells in a liner fashion (perfect line as if the plate was scratched there and they like it).

Hi Adrian, 

It's funny you say that we had the same problem with the falcon plates, I found the cells would do the same thing and line up as if it were scratched and generally they didn't really like it too much. Lucky they weren't actually our supplier but I borrowed a pack off another lab when I ran out. Always thought it was strange.The cells will grow and differentiate in clumps they like to be near their friends in the early days then by day 6 you should have a near monolayer of cells attached to the plate. 

We use the Nunclon range of sterile tissue culture plates - https://webshop.fishersci.com/insight2_uk/getProduct.do?productCode=10098720 I normally use the 144mmx20mm dishes cat number 168381. I've also used the 60mm dishes and the 92mm and they all work fine. You just have to get the seeding density correct for them.  I also use the corning 6 well tissue culture treated plates for my cell signalling work and the cells are happy on them - I'm lysing them straight in the dish with RIPA buffer so I don't want them coming off. I'll have to admit I am not a huge fan of BDs culture plastics, I've had lots of trouble with their T75 flasks when I was an undergrad and every time I end up having to use them something goes wrong. 

This is pretty amazing. I'm glad we put it here for others to see.

I only find the Falcon stuff to be good for fluorescence, as the corning flasks have a high background.

the ones you said you use say " with cell culture surface" - the description suggests that they are the same as "TC treated".

"Has a Nunclon™ Delta surface treatment which is a proprietary surface modification that promotes maximum adhesion for a broad range of cell types"

Did you mean a different Cat number? If so, which one?

I'll keep that in mind when I come to do my fluorescence work. I have so much IHC and microscopy work to do this year it's insane. I think they have different cat numbers on the UK Fisher site for some reason.

The ones I use are:

Nunc™ 249964 Plastic Petri Dish With Lid, O.D. x Height: 140 x 20mm, Sterile (Case of 80)

Nunc polystyrene (PS) petri dish; used for the culturing of fungi, bacteria and other microorganismsTotal volume: 250mL; suggested working volume: 35mL; culture area: 145cm²Material: polystyrene (PS); external dimensions (including lid): 140 x 20mm; internal dimensions: 136 x 18mmCompatible with automated systems and performs well in automatic dispensers due to complete flatness and uniform heightComes with a lid; vented; sterile10/Pack; 80/CaseManufacturer: Thermo Scientific Nunc®Manufacturer Part No: 249964

Very interesting discussion, thanks a lot. I have also had some kind of "lines" with BMDM on TC BD flasks, but I thought it was coming from the cells!

Did you ever try to maintain the BMDM in culture for long periods? Do they proliferate?

Hi, thank you for your input! Just to confirm, this protocol works for performing a FACS analysis of the macrophages?

Many thanks!

Hello Adrian, 

I am working on the macrophages. I really find this discussion very helpful. I would like to ask what is the best petri dish for the macrophages. I am really confused about the size and the brand. The plates samantha mentioned are 140*20 but the protocol says it should be 10 cm. I have been trying to seed the cells on 6 well plate(corning) with starting 4*106 per well in 2 ml. I find they are small in number at the end of the experiment.  Could you please let me know your opinion? 

Thanks

Hi All

very interesting information. just to add I agree to all above discussions and points. yes, the cold media is very important when harvesting the primary macrophages. regarding the plate I used the corning petri dishes from sigma cat# 430293 100mmx20mm and the cells will adhere strongly and hard to come off, therefore adding the cold media will help in de-attaching   cells with gentil scrape. also used the non tissue culture  one and they differ in the attaching, as the cells will be loosly attached  and also I noticed that the morphology of the cells will be different. but always doin FACs for surface marker F4/80 and CD11b will be the point when you know whether the macrophages are ok or not.

Thanks

Hi,

Please see the attached detailed protocols adapted from macrophage guru Siamon Gordon (one of my former PI's did his PostDoc there). This method works really well and I get around 30-40 million BMDM per mouse. Try not to disturb the cells too much. The only thing we do after seeding them is adding medium on day 4. We harvest on day 8 using a lidocaine solution which gently detaches the cells from the Greiner plates we use.

As for you polarization questions:

M1 --> 10ng/ml IFNy+ 100ng/ml LPS

M2a --> 10ng/ml IL-4 OR IL-13

M2c --> 10ng/ml IL-10

We do see the inflammatory M1 spontaneously switching to M2 after 24 hours of LPS stimulation. The reason for this was that they started producing IL-10 themselves in order to switch to a M2c tissue repair phenotype. These cells are very plastic and will have a different phenotype in a changed environment.

Hope this helps!

Hi,

I usually added the stimulating factors as stated by Anaouk A J Hamers. You can also check the protocol followed by the authors in Mary Jo Wick's research articles. I always used that protocol and it always worked for me.