Do you culture for 6 or 7 days, do you plate a certain number of cells on Petri dishes, do you wash the media on day 3?
Normally you would use media with M-CSF in it to culture BMDMs, in my lab we use L929 conditioned media. L929s are fibroblasts that secrete huge amounts of M-CSF and you just culture the L929s in flasks for 2 weeks, passing them as and when they need to be, you can normally judge it well by eye. When they look so squished just split them or move them up to a bigger flask. The media will contain M-CSF so you can just collect it, filter it and ass 20% of this to a bottle of DMEM high glucose + L-glutamine media containing 10% FCS, 5u/ml and Pen/strep, and it works well for us.
I isolate my bone marrow from mice and seed them in 10cm Petri dishes in a 20ml volume - we don't normally count them and seed them at specific densities - and incubate at 37C 5% CO2 and high humidity - and passage them on days 3 and 5. To passage the cells you remove 15 ml of the media and spin it down in a falcon, re suspend in 15 ml of fresh medium and place back in the dish, your total volume should be 20ml agian. Most of the macrophages will have adhered to the bottom of the petri dish, though some of the cells I work with have a deletion that makes them less adherent so I spin them down just to make sure I don't loose a lot of the cells.
The L929 conditioned medium is the one I currently use. However, use of the BMDMs in stimulation assays results in high variance of TNFa production and I was wondering whether the differentiation step could potentially induce slightly differently functional macrophages. This is why I thought that maybe another protocol would cause less variance in my system.
Macrophages are hetrogenious they just are, the whole M1/M2 phenotypes are sliding scales of potential rather than distinct subsets of a cell group and unfortunately the variation in their functionality is a consequence of that. I haven't seen any major variance in TNFa myself what are you doing in your stimulation assays?
You may want to screen your source of FCS. There can be tremendous lot by lot variability due to presence of LPS and other factors.
@ Samantha , it s a standard stimulation assay with LPS/IFN and from day to day absolute numbers of TNF vary. @ David, FCS has come from the same batch so far but I ll keep this in mind.
That is strange Theodore, as far as I know none of us in the lab have been seeing any kinda of variation in TNFa production. I would check your FCS as David advised if I've had any problems with cell culture that is normally the source. What are you using to measure TNFa? Intracellular facs or elisa or?
We typically plate 10x10^6 cells on a 150 mm petri dish. On Day 3, we refresh the media/MCSF and we typically stimulate the cells on D6 before harvesting on D7. I have heard a variety of opinions on which day to harvest the cells (anywhere from D5 to D7), but make sure to keep the day of harvest consistent between experiments.
I remarkable protocol can be found in the following reference:
Francke A, Herold J, Weinert S, Strasser RH, Braun-Dullaeus RC. 2011. Generation of mature murine monocytes from heterogeneous bone marrow and description of their properties. J Histochem Cytochem 59(9): 813-825. doi: 10.1369/0022155411416007.
I used L929 conditioned medium, plate 8*10^6 cells/10 ml. 6 days are enough..I throw away the medium and collect the cells with warmed macs buffer (pbs 1x edta 2mM fbs 2%) - 2ml per dish/ twice
We are also having some troubles in the same line (basal IL6 expression by Q-PCR in steady-state BMDMs from time to time). We are testing the possibility of minimun amounts of LPS in the intitute's prepared PBS that could account for this variability. BMDMs are exquisitive in their capacity of sensing endotoxin.
I actually culture for 5 days, replate them in 12 well plates overnight and infect or treat on day 6. I also use L929 conditioned medium and I have noticed some lot-to-lot variation. This is probably due to slightly different growth conditions of the L929 cells when making the medium. One concern is that you may have micoplasma contamination of the L929 cells. We frequently test our cells to make sure there is nothing else growing in them. One alternative is to use M-CSF instead of L929 cell medium as it is more consistant, but my experience is that you get fewer cells with M-CSF alone. The L929 cells must secrete other growth factors in addition to M-CSF that help with macrophage differentiation.
I do the same a Samantha Le Sommer. But make sure you use PETRI DISHES that are NOT (!) TISSUE CULTURE TREATED (i.e. the ones you use for agar plates to grow bacteria), as she pointed out! Otherwise you will never be able to remove the macrophages from the plates to split them.
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