I am trying to isolate and culture monocytes from canine bone marrow with the goal to differentiate them into macrophages. I am flushing the shaft of femurs from recently euthanized dogs (I know this is less than ideal) with essentially the same technique that is used in mice, removing the ends of the bone and flushing the medullary cavity with cold PBS.
After around 7 days, my flasks contain numerous fibroblasts that are outgrowing the few macrophages present. I hope to use canine GM-CSF in the future to try and get a higher yield of macrophages, but is there anything I can do to reduce or eliminate the fibroblast contamination? I haven't been able to find this issue addressed in any of the literature with mouse BMMCs.
Also, if anyone has experience culturing BMMCs from DOGs, I would welcome any advice! Thanks!
Hello Lauren.
I put the suggestions I did in a similar question.
Mixed cell population could be further purified by a 'negative selection method' as follows;
1) Set 20~30% confluency of the mixed cells (just ~two-folds of cell number as in routine subculture in ~8x 60mm dishes (if the cells are scarce, you could use 30mm dishes or 24-well dish (The best results are obtained from 60mm dishes due to larger area, avoiding dead ends of the dish walls).
2) Mark the time below & back them to incubator.
3) Set the times for 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4h (prepare dishes at different time points so that you can observe all of them simultaneously at the end of the experiment) in incubator.
4) Check the cell attachments and suspended cells by a gentle tilting.
5) The attached cells first would be fibroblasts which you do not want.
6) Gently suck the suspended cells after 2~3 gentle whirlings, that is what you want, tumor cells, for further your proper experiment or assays.
7) You can determine now how long you have to leave for highest cell purity.
8) This 'negative selection' or 'differential cell attachment selection' were coined in my lab, and works fine in our hands while we dealt with mixed cell population.
https://www.researchgate.net/post/Are_there_any_other_methods_of_determining_yield_efficiency_of_MACS_magnetic_activated_cell_sorting_separated_cells
Best wishes.
I would check the cells after 4 hours and take only the non attached ones...replate them...wait 4 hours...do the process again...you should remove most of the fibroblasts in this way
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs