小鼠骨髓细胞分离 Bone Marrow Aspirations in Mice.docx

Bone Marrow Aspirations in MiceI will be preforming bone marrow aspirations on a line of transgenics with the intent of culturing the cells. My lab has had prior issues with this, not getting the same yield of cells that others have observed. Previously, we have extracted the femur of the mice, cleaned the bone and then flushed the marrow out with PBS in an aspirating needle.Does anyone have any tips for this? As we all know, its the small things that tend to count.Thanks!TOPICS Bone Marrow Transplantation Bone Marrow Examination Bone MarrowSep 18, 2012Share1/0ALL ANSWERS(10)Martijn H. Brugman Leiden University Medical CentreHi Ashtyn, Ive tried BM aspiration in mice before, but found it rather difficult. A colleague of mine, who is more skilled at it, uses the technique that is described in Verlinden, Experimental Hematology 1998. Perhaps this is useful. From your question, im not quite sure, but are you talking about bone marrow isolation after you euthanized the mice?Sep 19, 2012Christoph Metzendorf Universitt HeidelbergHi Ashtyn,we routinely do this to make bone marrow derived macrophages. I see some fluctuations in cell counts (reaching from 2.25x106 to 3.5x106 cells/mL in 10mL of buffer) but generally they are no problem as we count cells and then seed the same number of cells per well. We extract BM from both femurs and tibias of each mouse.One thing that seems to affect cell counts are age of mice and strain.If you are interested in a more detailed protocol, I uploaded it here.o BMDM prep and culture.pdfSep 19, 2012Hendrik Gremmels University Medical Center UtrechtHi,The protocol supplied by Christoph Metzendorf is very similar to what we are doing. There are some small variations across protocols: some people use RPMI for flushing the bones with the idea that it leads to higher viability. It is (slightly) more expensive however and you have less visual feedback of the marrow clearing up if its got phenol red in it.For cutting up the bones we use a surgical wire cutter, so you can cut right through the heads and waste as little as possible. You can do more bones if you need more material, you should be getting around 20M cells from the two femurs, another 10 M from the tibia and maybe 5M from the humeri although mileage may vary.Of course this is al terminal for the donor mouse. Im not sure if there are methods that preserve the donor.- HendrikSep 19, 2012Ashtyn Zinn University of ToledoThank you so much for your input! Im picking up a copy of the 1998 Experimental Hematology text today and Ive looked over your protocol, Christoph. There are some slight variations, like you said Hendrik, we have been using PBS to flush and our cutting techniques are slightly different. I think I will try the HBSS as well as a surgical wire cutter this time.Thanks again all!Sep 19, 2012Milivoj Boranic Ruer Bokovi InstituteHi, There are some tricks. 1. Disssect femur and tibia carefully, do not cut or break head of femu, try to exarticulate it from acetabulum . 2. Du not cut distal end of femur, try gently to dissociate epiphysis from femoral shaft. If properly done, the shaft and epiphysis dissociate at the epipyseal cartilage (the line of bone growth). You will see four nodules at the end of femoral shaft. Insert a CURVED needle #20 and force the marrow out. If you do that with submersion into the fluid (eg. PBS), the yield is better. 3. Cut tibia at both ends and flush the marrow out. With tjis technique you may get as much as 10 million cells per mouse.Good luck!M. BoranicSep 19, 2012Martijn H. Brugman Leiden University Medical CentreI think all the flushing methods above should work fine. If you are interested in slightly higher yields, we also take out femur and tibiae (and possibly sternum), with heads and all, like Milivoj says, remove the muscle tissue with a sterile cloth. I then fragment the bones coarsely with scissors and crush them in 3 ml RPMI in a mortar. Straining over a 70um filter afterwards removes most of the debri. A second go at crushing in another 3 ml RPMI gives a slightly higher yield. For 2 tibiae, 2 femurs and sternum, I typically get 35-45 million cells per mouse.Sep 20, 2012Sophie Acton University College LondonYou can use both the femur and the tibia, and we had good yields using cold RPMI with 1% FBS. Dont spin the cells too hard eith