I would like to perform CFU-F crystal violet staining for rabbit bone marrow derived MSCs, but I can't seem to figure out the seeding densities for them. I isolated fresh bone marrow (1ml/rabbit) and obtained MNCs via density gradient cell separation using Ficoll. These mononuclear cells were then plated at P0 and adherent cells (at P1) were then used to seed for CFU-F at 150-, 500- & 1000-cells in 10cm dishes (in triplicates). As no colonies were observed at the end of fourteen days for all three seeding densities, I concluded that the either starting cell numbers were too low or the 10cm dish was too big. The main constraint that I face is the low cell numbers that I obtain at P1. Also, does anyone else observe the change in cell morphology from spindle-shape to big spread-out spaceship looking cells at later passages? Can you kindly share your experiences/insights on this?
Hi Zophia,
Those large spaceship looking cells are macrophages. One thing I like to do when establishing MSC from human bone marrow is to deplete the macrophages form the bone marrow sample by using CD14 labeled magnetic beads. I then use the macrophage free cells to start my culture. When you harvest your P0 cells how confluent are the cells? Depending on the size of the culture vessel, I normally let them get established (`85% confluent). Then I spilt then for P1 cells. I also due the CFU-F assay in a 6-well plate.
Good luck!
Thank you so much Daniel!
I finally got the CFU-F to work so I am relieved.
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