I have directly cocultured macrophages and MSCs fro 48 hours and now I want to separate the coculture with CD90 antibody-coated magnetic Dynabeads to separate the cells by magnetic force.
I have tried a bead/cell ratio of 6 beads per cell, which did not work properly after analysis by flow cytometry.
When I search for papers doing the same, the concentrations are not listed.
Can anybody help?
The optimal number of Dynabeads per cell for separating mesenchymal stem cells (MSCs) from macrophages can vary depending on several factors such as the sample size, the purity of the starting cell population, and the desired yield. In general, a range of 1-2 Dynabeads per cell is often used.
Hi,
Bead concentration
in general, a high cell yield is generally obtained using a concentration of Dynabeads™ of 1x10e7 Dynabeads™/mL or higher in the cell separation step. An initial concentration of Dynabeads™ giving a bead to target cell ratio of between 4:1 and 10:1 is generally optimal for the isolation. However for rare cells (e.g. stem cells from BM or leukophoresis) the cell concentration could be increased up to 1 × 10e8 cells/mL.
Mixing
Also important is mixing to avoid beads to settle (tilting and rotation).
Direct vs Indirect isolation
the indirect method may be considered as it is in general more efficient in removing target cells from a suspension.
- Use the indirect technique when: A cocktail of mouse monoclonal antibodies is used to deplete several cell types simultaneously (for this it is recommended to use MNC as a starting sample to remove erythrocytes, platelets and granulocytes), very high depletion efficiency is required, the affinities of mouse antibodies are low, the cells express low number of target antigens or the direct technique gives unsatisfactory purity.
- Use the direct technique when: The affinity of the primary antibody is high, the cells express a high number of target antigens or to make a larger stock preparation of primary coated Dynabeads™ (will generally have the same shelf life as the stated on the Dynabeads™ vial
kind regards
ketil
Ketil Winther Pedersen thank you for the tips! We went with the direct technique as the affinity of our primary antibodies is pretty high and CD90 is widely expressed on the MSC's cell surface.
We now tried three different bead-to-cell ratios (5:1, 10:1, 20:1). We managed to retrieve a cell population where the 20:1 ratio was the best in separating the coculture. Although, it only halved the population of the MSCs from macrophages (10% of cell population are still MSCs after separation).
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