Hi, the standard way is to isolate bone marrow cells from femurs. You can find all the details of the protocol in Current Protocols. See the link: http://onlinelibrary.wiley.com/doi/10.1002/0471142735.im1401s11/abstract

Hi, just go with Femurs. I always cut the bone on hip joint and get the entire leg. Clear off all the skin very carefully and then cut the bone with nice pair of scissors on the knee joint. Keep a 10 ml syringe with 26G needle ready with RPMI (and not PBS). Now hold the femur with a pair of forceps and insert your syringe in it and flush out the entire 10 ml thru it in a petri dish (containing media). You will see that the entire bone will turn white as all the bone marrow has been flushed out. Pipette up and down vigorously to  disintegrate the bone marrow cells in the petri dish. Collect the entire media in a 50 ml conical tube and centrifuge it at about 300-400 g at 4deg (preferably) for 5 mins. Now you can resuspend them in a desired volume and plate them on surface of your choice and keep them in 5%CO2 incubator for 7days after which you will see Macs. Usually by this method you should be able to get about 50 million cells per mouse (ie two femurs).

Thank you both!

Deepa, regarding this procedure, I don't have to differenciate between Macrophages and any other cell during the culture of the whole mount of the bone marrow? Just doing that I would have the entire population of macrophages?

The BMDM have to be stimulated in culture. Add CSFs or IL-3. Then you can differenciated them because they adhere to the dishes.

Hi,

I use both the femurs and tibia for bone marrow collection from mice. From a healthy 4month old mouse I normally get about 30x10^6 cells. Dissect out the leg from the mouse into media and place on ice, I normally transfer the legs into our TC hood and then further remove all the muscle and other crap to get the bones, you will see they are a weird red colour that's the bone barrow inside the bone. Hold the bone in forceps and cut off the top and bottom of the bone. Flush each bone with 10ml of PBS into a 50ml falcon through a cell strainer. Do this for each bone, you will end up with 40ml of PBS containing bone marrow cells. Some of the bone marrow will come out in big clumps I normally use a sterile pasture pipette to push them through and wash out the filter to make sure that you have all the cells.  Repeat for each mouse.  While your doing each mouse keep the cells on ice.

Once all the bones have been done spin down the cells in centrifuge, I spin them down at 1800rpm for 7 mins in our centrifuge I'm sorry I don't know the g off by heart! You will see a large red pelt, the bone marrow contains lots of red blood cells dependent on your downstream application you usually want to get rid off them. Resuspend the cells in 1ml of Sigma Red Blood Cell Lysing Buffer and incubate for 10 mins at room temperature. Add 10ml of PBS to each tube and spin down again.  I find if I'm doing any downstream flow cytometry or western blotting getting rid of the RBC is a must or the staining and blotting can be dirty.

I normally then resuspend my cells in 1ml of complete media then I count them. I seed my bone marrow cells at 5 x 10^6 in a 10cm bacteriological petri dish as I use them for flow cytometry normally and harvesting macrophages from tissue culture treated places is a nightmare. Macrophages adhere well so a TC treated surface sometimes isn't the best idea. For my Macrophages I use 20% conditioned L929 media I have a protocol for the generation of this if you want it, or you can use 20ng/ml of recombinant MCSF. I find that the L929 media works better I get a higher yield of cells and better differentiation plus it's much cheaper too.

Seed 5 x 10^6 cells in a 10cm in 10ml of complete media, then top up to 20ml the next morning. This gives the cells a better change to settle onto the bottom and to start differentiating. On day 4 of culture remove 10ml of media and refresh with 10ml of fresh media. On day 7 your cells are ready for stimulation / experimental use.

Hi

You will have to add MCSF or L929 conditioned media to differentiate them to macrophages. For differentiating them to dendritic cells you can use GM-CSF and IL4 or FLT3 ligand. If I may ask, what do you want to use them for?

Samantha Le Sommer,

Can you please tell me how much do you get on day 7 after seeding 5x10^6 cells?

You normally get out between 3 - 4 x 10^6 cells. Not all bone marrow precursor cells will differentiate, and those which do not differentiate are normally eaten by macrophages so there is some cell loss. I normally harvest cells with stem cell accutase if I need them live for down stream applications, or scraping in lysis buffer for protein / RNA 

Dear Samantha,

Thank you for the reply.

I'm working on BMDMs and I'm trying to find a protocol which give an optimal yield of macrophages on  day 7.

I seed my cells in 10 cm bacteriological petri dish at 5x10^6 with 10 ng/ml of recombinant M-CSF with 10% FBS + 55 μM 2-mercaptoethanol, 100 U/ml Penicillin, 100 μg/ml Streptomycine and 292 μg/ml Glutamine. I got around 1x10^6 cells.

Maybe the concentration of M-CSF was insufficient, so I tried with 100 ng/ml. I got around 2,5x10^6 cells.

Now, I'm getting confused why my cells don't differentiate..?

Hi Donald, there could be a few reasons why you might need to do a bit of trouble shooting;

1 - there may not be enough glucose and amino acids in your media, I also supplement my media with Non Essential Amino Acids - we buy in 100X stock from Gibco and then add it to the media so I'm not sure about the exact concentration in the end.  Is there sodium pyruvate in your media as well? If not we normally supplement that in too at 1mM just to give some extra buffering. 

My media recipe is here;

• Gibco DMEM High Glucose High L-Glutamine (500ml)
• 10% Foetal Calf Serum 
• 20% L929 conditioned media OR 20ng/ml MCSF
• 100U/ml penicillin & 100mg/ml Streptomycin 
• 1 x Non Essential Amino Acid 
• 1mM Sodium Pyruvate 
• 0.25mM Beta-Mercaptoethanol 

2 - when are you changing the media? I normally seed the sells in 10ml of media to start with on day 1 then leave them over night and gently add in another 10ml the next morning to bring the total volume up to 20ml.  On day 4 remove half the media and add in 10ml fresh media, then do the same on day 6. Your cells should be ready to use day 7. I have found removing all the media hampers differentiation, they must be secreting things helping with the differentiation. 

3 - I've used MCSF in the past, I used 20ng/ml, we use L929 conditioned media in the lab I'm in now and I've found I get a much higher yield and more happy cells. If you can get ahold of a vial of these cells in the long run it will be a lot cheaper and personally I find the results are better.

4 - How are you harvesting your cells? The process your using may be inadvertently damaging cells or killing them, if you are trying to harvest to count scraping them isn't the best method to use and typically results in 50% death of your culture with primary BMDM 

Cutting the femurs of mice (2 back legs) and flushing out the bonemarrows using media (DMEM or RPMI) gives you about 15-20000 cells per mice on average. To check for purity of your macrophages, 7 days post differentiation flow cyt for CD11b and F4/80, for the double positive population. If you need the detailed protocols, let me know.

Hi Samantha,

Thank you.

1- I use RPMI-1640 medium for the culture (with 2000 mg/ml D-Glucose). I do not use sodium pyruvate and non-essential aminoacid. I may try it.

2- I change the medium on day 2,4 and 6. I remove 7 ml of old medium to add the same volume of fresh medium. The final volume is always 10 ml.

3- As using cell scraper damage the cells and addition of trypsin stress them, I harvest my cells by rincing the dish wih pre-incubated PBS. The pressure applied when flushing PBS on the dish detach the cells. When finished, I always make sure there is no attached cell on the dish.

Hi Sreeram,

Thank you for your comment.

I'd be grateful if  I can have a look to your protocol.

Hi Samantha,

Thanks for your protocol. Could you please tell how you harvest your cells from the dishes for further seeding on plates?

Hi Nizar,

I  use StemPro Accutase ( https://www.thermofisher.com/uk/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html ) for getting the cells off the plate. Make sure you grow them on sterile bacteriological plates, if you use tissue culture treated surfaces they aint coming off at all. 

Remove the media from the place and discard, then gently wash the plate with 10ml of cold PBS and place this in a falcon tube. Add 2ml of accutase and incubate room tempreture for 5 - 20 minutes depending on how well the cells are stuck down and the density, then wash again with 10ml of cold PBS. Cells should lift stright off the plate. Centrifuge them and then re-suspend in whatever for your downstream applications. I've done both flwo cytometry and adhesion assays down stream of this and it doesn't affect of any of the markers or functions of the cells from my experience. 

Just a tip as well, dont heat the accutase, I've found it works more efficiently at room temperature or below!

Donald,

I've found the BMDM are quite metabolically active when they are differentiating so if there are shortages of amino acids in the media then it can be pretty damaging for them. Supplementing it may help. 

I seed my bone marrow in day 1 in 10ml of complete media, then add in another 10ml of media on day 2 to give a total of 20ml. On day 4 I remove 10ml and add in another 10ml of fresh media to bring the total back up to 20ml. You may be removing too much of the media and thus too much of the autocrine signalling molecules that are in the media. 

Your harvest sounds fine so its prob not that, I'd tweak your culture protocol a little, also when you're culturing them leave them alone. I know that sounds really counter intuative but the less you play around with them the better. I don't touch them between changing the media and moving the dishes around to take them out and look at them every day can upset them. As I tell my students "stick them at the back of the incubator and don't touch them, they wont work their magic if you are watching!"

:) 

Thanks alot Samantha :)

Hello Samantha, 

I have been trying to culture macrophages. I plate the BM cells on 6 well plate (corning) with 2*106/ml  with 2 ml per well and change 50% media on day 3 and then change to treatment with ifng and il-4. When i look at the cells under the microscope i find them very confluent giving impression they are at good numbers. I have found that the rna yield from the wells is very low.

Thanks.

 Hi Guys what concentration of beta mercaptoethanol you use in medium??

Hi Ahmed - Oh my gosh I am so sorry for the slow response I didn't realise you had asked a question, I'm so sorry! What RNA yield are you getting, I normally get about 40ug of RNA from a well. I've found low yields with mine as well and I found it was the lysis step that was responsible. We use TriZol in my lab and phenol-chloroform based RNA extraction. I used spin columns once but just had really bad results with it. I used 2ML of trizol per well and then followed the protocol from there. Just make sure you scrape the wells really well! 

Hi Daleep - I use 0.01mM of beta mercaptoethanol in my media :) 

Hello Samantha,

Thanks for your answer. It is ok. Surely you have a busy schedule. I had very low RNA yield like less than 1 ug. We use TriZol and phenol choloroform extraction. I use 1 ml per well. In the beginning, I add the trizol and pipette it few times and then continue with extraction which was was very low. I did scraping as you mentioned and the yield was still low. I don't know what is wrong. May be the the volume of trizol is responsible. Thanks 

Thanks a lot Samantha for the advices!

Two questions:-

1.  I usually take them on 6 th day of differentiation from 20% L929 media and plate them for experiment in 10% L929 condtioned media. What markers to use to check for differentiation of these macrophages at 6 or 7 days post-plating the bone marrow in L929 cnditioned media?

2. post-differentiation when BMDMs are plated for experiment how can I get them off from 6 well TC dishes for flow cytometry experiment?

Hello Alka,

Answering to your questions:

1.- I normally use CD11b and CD11c. I tend to obtain a < 90% of CD11b+CD11c-

2.- The best recommended protocol is using 2mM of EDTA, incubate them at least 5-10 minutes on the hood and bang the plate from time to time to lift them off. I normally start pipetting up and down to make it faster, but make it gently. 

Hi Alka,

Answers to your questions: 

1. I use F4/80 and Cd11b markers for checking differentiation quality. Usualy obtain 99-100% double positive population. 

2. Like Francisco said 2mM EDTA will work. But sometimes they get really sticky, and slight scraping may be required. 

Ok, thanks.

Hi,

This is the best protocol for BMDM.

Good luck!

Hi Francisco,

We puplished a little paper recently that describes transfection of pirmary macrophages but also describes very detailed the preparation of bone marrow-derived macrophages and peritoneal macrophages.

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

What procedures would you recommend for harvesting the cells at day 7? For Western Blot. How much lysis buffer would you add to each well? I found answers ranging from 100ul to 250ul to 500ul.

I have added 300-350uL of lysis buffer on Day7 of seeding 3x10^6 BMcells for BMDMs and it has worked.