What are Bmdm cells?
Bone marrow-derived macrophages (BMDM) are primary macrophages obtained by in vitro differentiation of bone marrow cells in the presence of macrophage colony-stimulating factor (M-CSF or CSF1). They are easy to obtain in high yields, can be stored by freezing, and can be obtained from genetically modified mice strains.
What is the meaning of transfection?
Broadly defined, transfection is the process of artificially introducing nucleic acids (DNA or RNA) into cells, utilizing means other than viral infection.
How do macrophages transfect?
For transfection the predifferentiated macrophages are detached enzymatically by Accutase I treatment. The transfection is performed using a Nucleofector 2b device for electroporation of the cells. After transfection, differentiation is continued for another 24 to 48 hr as required.
How do you get Bmdm?
Harvesting BMDMs Detach BMDMs by gently scraping with a rubber policeman. Collect BMDMs in 50 ml tubes and centrifuge at 450 x g for 10 min. Gently dissociate cell pellets in 20 ml complete DMEM. Count BMDMs in the presence of trypan blue (no more than 10% mortality should be observed).
How many Bmdm are in one mouse?
We normally differentiate our BMDM with M-CSF (10ng/ml) and we also get around 30 million per mouse THe floating cells are normal. We once tested L-929 and got much more cells, but they did not respond at all after bacterial infection or PAMP stimuli (with expection for cell death).
What is another word for transfection?
In this page you can discover 11 synonyms, antonyms, idiomatic expressions, and related words for transfection, like: immunoblotting, immunoprecipitation, antisense, rnai, immunostaining, transfected, oligonucleotides, , inactivation, sirna and mutagenesis.
How do you transfect cells with mRNA?
The mRNA transfection process is more simple: mRNA is directly delivered and expressed in the cytoplasm and thus does not require to cross the nuclear membrane. After delivery, it can immediately be translated into a protein in the cytoplasm.
What are monocytes and macrophages?
Monocytes and macrophages are very closely related cells with a few important distinctions and different use cases. Put simply, monocytes are macrophages in the blood; macrophages are monocytes in tissue.
How do monocytes convert to macrophages?
Monocytes can differentiate into inflammatory or anti-inflammatory subsets. Upon tissue damage or infection, monocytes are rapidly recruited to the tissue, where they can differentiate into tissue macrophages or dendritic cells.
What are the transfection rates of pm and BMDM?
With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.
Can you generate BMDM from frozen bone marrow?
Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM.
What are the steps to a successful BMDM culture?
There are several critical steps for successful BMDM culture: 1) maintaining a sterile, healthy culture; 2) bone marrow extraction needs to be efficient; 3) L929 supernatant quality is critical; 4) do not expose cells to the red blood cell lysis buffer for more than 2 min to avoid hematopoietic cell damage.
Is the BMDM selection difficult for sequencing analysis?
The BMDM selection could be a little bit difficult if I need large scale of cells for sequencing analysis for example, I suppose if I increase the dosage of the virus I will manage to get nice knocking down without puromycin selection. Does anyone have experience on that.. Join ResearchGate to ask questions, get input, and advance your work.