I have also worked with a protocol adapted from M. Lutz as referred to in answer 1 already. It worked very well and you can generate large amounts of DC. Depending on the rationale for your experiments, you should bear in mind that bone-marrow generated DC rather reflect only a certain subpopulation of DC (like inflammatory DCs in the lung). The exact protocol is described in our publication:

Clin Exp Allergy. 2011 Nov;41(11):1609-21. There you can also find references for the bmDC characteristics of lung inflammatory DC.

Hope this helps,

good luck with your work!

One useful tip: you can freeze bone marrow, make aliquots and use it to generate DCs at any time...

Hi Kimberly, i sent you our protocol via Research Gate messages! Good luck, best, Doris

Hi Kimberly, we use the "Lutz protocol" with great success. Check the paper J. of Immunological Methods, (223), Issue 1, 77–92. Lots a luck! Best, Cristina

You can check with my paper published on Cancer Immunology Immunotherapy in 2001 for the protocol.

This method is from one paper and I have used it. "The bones were placed in

70% ethanol for 2 min and subsequently washed

in PBS. Both bone ends were cut off and the marrow

was fl ushed out with RPMI 1640 medium

(Gibco, Grand Island, NY). The red cells were

lysed with ammonium chloride (0.45 M). The cells

were centrifuged for 10 min at 1,500 rpm and

2 × 105/ml cells were cultured in Petri dishes in

10 ml of RPMI1640 medium with 10% fetal bovine

serum (FBS); 2 mM L-glutamine, 100 U ml−1

penicillin and 100 mg ml−1 streptomycin (complete

medium) and supplemented with 5 × 10−5 M

2-mercaptoethanol and 15%–30% of mouse

granulocyte-macrophage colony-stimulating factor

(mGM-CSF)"

Hi Kimberly.

You've seen my answer to Shakti Singh's question "How many BM-DCs one mouse can yield?" (thanks for the vote). I hope you got all the information you need. If you want to discuss something else in particular you can email me. Good luck!

We had great success with this protocol as well.

Hi Kim

I have worked with a protocol for isolation of DCs . I have the detailed SOP as well. If you are interested I will send it to you via e-mail. Thanks.

Dear Kimberly,

Here is a link to a visual method. It´s perfect.

(P. Matheu, M., Sen, D., Cahalan , M. D., Parker , I. Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging. J. Vis. Exp. (17), e773, doi:10.3791/773 (2008)).

http://www.jove.com/video/773/generation-bone-marrow-derived-murine-dendritic-cells-for-use-2

Good luck!

Javier

Have any of you produced GM-CSF through CHO cell lines and if yes would you please send me its protocol as well as generating dendritic cells from mouse bone marrow using GM-CSF. Thanks