Yes, it is. Targets can be removed from the pluriBeads under physiological conditions at room temperature. This way, removal is gentle (low cell stress) and fast (10 minutes).
The obtained single cell suspension can be used for further downstream experiments such as FACS, cell culture experiments or nucleic acid isolation. All reagents necessary for removal are included in the kit.
Yes it is! Particularly cell lines used in cell culture experiments often have the ability to grow on different kinds of plastic surfaces without showing adverse effects. Cells attached to the beads can be transferred to tissue culture dishes and the cells will attach to the plastic surface over time, depending on their mobility characteristics.
If desired, the targets can also be detached from the beads using pluriSelects' detachment buffer and separation strainer (included in each pluriBead reagent kit).
Yes, it is. Particularly easy to educe nucleic acids from RNA/DNA of the cells separated with pluriBead. Following the isolation, target cells can be lysed directly on the strainer using commercially available lysis buffer according to the manufacturers’ instructions. Thus the detachment step can be spared which saves time and prevents cell stress.
The apoptosis rate is below 10%. This rate is achieved by low cell stress due to two inherent characteristics of the isolation kit: natural environment of the cells (by working with unprocessed sample materials) and minimal handling time.
Some work steps are obsolete (e.g. cooling or changes to the cells' environment by Ficoll™ density centrifugation), others are reduced to a minimum (e.g. pipetting and centrifugation). This results in minimal mechanical stress and the isolation duration is kept to a minimum. Thus, the isolated cells can easily recover in cell culture medium shortly after the isolation or can be used in other assays.
In contrast to other available systems, pluriBead works without unnecessary pipetting steps. This does not only reduce the loss of precious cells or proteins but also lowers the apoptosis rate. According to the standard protocol, an average 80% of the targets are retained and isolated.
S-Beads: 40µl s-pluriBead suspension have the capability to catch 10^6 targets in 1ml whole blood.
M-Beads: 100µl m-pluriBead suspension have the capability to catch 3x10^6 targets in 1ml whole blood.
However, the number of targets that pluriBead particles will actually catch depends on the amount of target cells in the sample material, the density of receptors on target cells and optimal mixing of the particles in the sample.
The initial cell distribution (lymphocyte/monocyte/granulocyte) can be determined by using a hemocytometer or with a counting chamber and Türk‘scher solution.
Please keep in mind that the distribution of cells may differ between different people. For rare cells, lower yields are a consequence of the small population of targets in the sample material.
The average purity of isolated cells achieved with the pluriSelect technology is 95%.
We recommend to isolate your targets (cells, proteins etc.) at room temperature or even at 37°C. The advantage is that the cells are not subjected to great stress because they stay in their usual environment. This way, target cell vitality will be above 90%. It is not necessary to cool down your sample to 4°C. In contrary, if you cooled the sample its’ viscosity would change and the binding capacity of your targets to the beads would be reduced.
The terms PBMC and buffy coat are sometimes used like synonyms. But there is a difference between them: PBMCs are won by density centrifugation with a medium. They are pure leukocyte suspensions, without granulocytes. Buffy Coat however, is won by centrifugation with a filter, so that most erythrocytes are extracted. That means, in a buffy coat there are still granulocytes, lymphocytes and some erythrocytes included. Because of the different composition of these two materials, we recommend different cell separation protocols:
- PBMCs require needs an incubation buffer during the separation
- Buffy coat kits do contain stabilization buffer and extra strainers for pre-filtering of the sample.
Yes. You do not need to prepare your sample material in any way (Ficoll gradient or others), but work directly with your sample.
None. pluriBead has been especially developed to isolate cells or proteins directly from whole blood or buffy coat. Isolation beads are mixed directly with the blood sample and incubated a standard tube. The main determinant for the target specificity is the antibody bound to the surface of the separation bead. Other molecules in the heterogeneous sample do not interfere with the binding reaction. So, the removal of erythrocytes by density gradient centrifugation or erythrolysis prior to using the pluriBead cell isolation kit is not necessary. In fact, the viscosity of whole blood reduces cell stress and consequently minimizes the apoptosis rate of the isolated cells.
pluriBead is optimized for working with whole blood, buffy coat and tissue/cell culture.
The rounds per minute (rpm) setting of the horizontal roller mixer, or pluriPlix respectively, can be used quite variable. However, 8-15 rpm is optimal for a complete mixing of the sample with the separation beads. The rotation speed should not fall below 5 rpm and should not greatly exceed the optimal settings. Otherwise, beads would settle or flow rates would increase. Both processes result in reduced bead-target binding reactions and increased cell stress.
pluriBead isolation technology requires only minimal extra lab equipment. As sample preparation is not required, centrifugation steps are unnecessary. So, all you need is standard equipment such as sterile pipettes, tubes, gloves and an appropriate mixer that keeps the beads from settling and allows for a rotation speed of 8- 15 rpm (e.g. pluriPlix or a fixed angle or tilting horizontal roller mixer).