Hi Steven,

I've only been working with L929 cells for a year now, so I can't give you the ultimate answer if a lot of passages are bad for the MCSF production. However you anyway have to test your conditioned media prior usage (% in Media, to get the same amount of macrophages as before) for every batch. At least thats what I do.

I also try to not use them to "long" however. Freeze a lot of aliquots in the beginning maybe.

As for the protocol- there are a lot of different ones available. I was told to first grow the cells in full media (DMEM, Glu, AB, 10%FBS) until confluent then split 1:9 in starving media (w/o FBS) and after 9 days collect the conditioned supernatant, sterile filter and store @-80°C.

However I prefer to starve them now when they're in the log phase: split them in full medium to however many plates you want/need - grow until 60-70% confluent - wash well and starve for 9-11 days. Also I strongly recommend NOT TO USE ANTIBIOTICS!!!

Its better to see an infection and discard the plate (and even if bacteria growth is inhibited/ killed their products will still activate your macrophages)

good luck!

Hi Steven,

we used fresh populations. Then we expanded them and plated them with 1 Mio cells in 10 cm dishes using media with 5 % FCS. After 6 to 7 days you can harvest the supernatant.

If you use FCS free medium, you have to start with more cells (like 2 to 3 Mio.).

Be sure the cells are free of mycoplasmen.

Good luck.

@ Monika.

Hi.

It may be a while since your last comment but maybe you can give me some advice here.

What was your experience with "not using antibiotics".

Did you see contamination very often, considering that flushing the bone marrow is not sterile.

Many Thanks in advance, Timo

Hi Timo,

No thats not what I meant - I am using antibiotics when I differentiate the BM, although I actually think that it should work fine also without. But I dont use ABs for the L929 cells, because when you have a lot of dishes (I use like 42-44 the 15cm for 1liter) and you need to wash them all with PBS things can happen. 

Also its my test for Interns if they can work sterile in the cell culture ;-) So its also for my own sanity that I dont have to triple check every plate before considering it to be bacteria free - you see a contamination CLEARLY if you have one!!

If you use FCS like Birgit make sure you use the same as for the MACs (low endotoxin).

All the best,

Moni

Hi Monika, 

I just tried out your method and was just wondering how you calculate your %. I've got some L929 cells that have been in DMEM with only L-glu, no antibiotics and no FCS for 12 days (we couldn't do 11 as I was away yesterday). They still seem to be perfectly happy and adherent (they're only 3rd passage) and they're in big culture flasks with around 150ml of media in each. We just did an ELISA to determine the concentration of M-CSF and it came out as about 1ng/ml. I've done a load of reading into concentration needed for macrophage differentiation and it seems to be that around 10ng/ml is the usual concentration used. How much M-CSF do you normally get? I'm a a bit of a loss about how to get to a working concentration of 10ng/ml when my totally undiluted L929 supernatant is only 1ng/ml.. I could leave the cells for another week in the incubator, currently there is no colour change in the DMEM even though they're very confluent (and beginning to pile up)! 

Any advice would be greatly appreciated!

Thanks a load,

Charly

Please can you tell me the difference between L929 and rM-CSF according to your experience ? are there other cell lines for Bone marrow derived macrophage ? 

Thank you

Hi Charly,

I usually just "test" it in terms of - how many BMDMs do I get after differenciation?  I just compare it to the last batch. I get more or less 10 confluent plates after 7 days (30-40x10^6 cells). 

I seed the BM on 5 petri dishes (10cm) after flushing femurs and tibias on day 1. Then I wash the plates on day 4 (to get rid of other cells) ad fresh media (DMEM, 10% low endotoxin FBS, Gln, AB, 50nM ß-mercaptoethanol and 10-20% L929 SN) and split the cells 1:2. Then I wash and harvest them on day 7.

Whats the growth area of your flask? I use 15cm dishes with 30ml of media, although now that you said it flasks would be much easier ;-)

But simply try your SN for in various concentrations!

@Dr DG

well shure its nicer and less work to use recombinant mcsf, but the other way is cheaper. I'm not really experienced with cell lines, there are a couple of monocytic cell lines though (like RAW, U937,..) .

Thank you Monika,

i also found some people raise a question about the gender of the mouse (male or female) is important in macrophages isolation (they were talking about peritoneal macrophages)! does it really matter and why ? 

Dear Dr DG,

That I really don't know! But I do guess that certain hormone levels might influence the populations quite a bit?! I just know that in a lot of studies gender matched mice are used for in vivo experiments,  as the variance is lower. So it must be hormones, right? 

Please let me know if you if you find out more!

Dear Monika,

I've found this paper which talks about this issue,

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1231091/

They mentioned that sex hormones affect the expression of many cytokines. For example, Estrogen down-regulate INF gamma and alpha nut up-regulate IL-10 which is suppressor for Th1, macrophage functions and T cell, so making the females are more susceptible to infection in some disease.However, in other studies, female mice are more resistant!

Also, you can go through this recent paper: http://www.bloodjournal.org/content/bloodjournal/118/22/5918.full.pdf?sso-checked=true

Can anyone tell me what kind of L929 cells

and what field of works could employ them?

 For 500 ml combine:  

100 ml   Heat inactivated fetal bovine serum 

5ml    Q (100 X Glutamine from freezer) 

5 ml    Pen/Strep (100 X)

150ml     L cell culture supernatant 

5ml     Pyruvate

DMEM   up to 500ml

Hi Sorocha,

Grow L929 cells, till con-fluency. Let the media stay for 4-5 days, centrifuge the media at 500g for 5 mins, filter with 0.2 micron filter and store it in -20 as one stores FBS/FCS. Use 20% in your media for growing macrophages. 

@Kajal Gupta Does your cells start looking stressed on keeping them for 4-5 days post confluency?

Hi Steven,

I have made many batches of LCM and have attached a detailed protocol for you. Do make sure you get the DMEM with pyruvate added in. We typically leave the cells for 10-12 days before harvesting the LCM. I have also attached a BMDM maturation protocol (adapted from Siamon Gordon).

Good luck!