FAQ

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).

• Consumables: room temperature for 12 months
• detachment buffer: -20°C for 6 months
• all other buffers: 4-8°C for 6 months
• pluriBead Suspension: 4-8°C for 6 months

pluriBead can be used in samples from 0.3 to 45 ml.

	S-pluriBead	M-pluriBead
Size	30µm	60µm
Maximum Target Cells Per Separation	10^7	5*10^7
pluriBead Suspension	2ml	2ml
Maximum Bead Suspension Per pluriStrainer	400µl	1000µl
Sample Material	Whole Blood, Tissue, PBMC, Cell Culture	Buffy Coat, Whole Blood, Tissue, PBMC, Cell Culture
Recommended Application	small number of targets in large sample volume and Circulating Tumor Cells (CTC)	large number of targets in a samples (e. g. Buffy Coat)
Minimum Sample Volume	200µl	500µl

S-beads: add 40µl S-pluriBead suspension per 1ml whole blood (approx. 10^6 targets)
M-beads: add 100µl M- pluriBead suspension per 1ml whole blood (approx. 3-5*10^6 targets)

Thirty minutes. Cell separation with pluriBead works faster than other available systems. For example, the separation of CD3(+) cells from whole blood is completed in 30 minutes. The reduced separation time is mainly achieved because common sample preparation steps such as Ficoll™ gradient density centrifugation are not necessary. Only three simple steps are to be performed until the isolation is completed:

1. mixing of sample and isolation beads
2. incubation at room temperature
3. separation of targets using the pluriStrainers and subsequent rinsing of the targets with buffer or medium. At this point your cells are separated from the sample and ready for RNA/protein isolation (e.g. microarray , qPCR).

   If bead removal is necessary, two very short steps are to follow the above procedure:

4. Incubation of the targets with removal buffer to detach the cells from the beads
5. Washing off the cells into tissue culture flasks or lysing the cells for RNA / protein analysis assays.

The total isolation time including the cell removal steps is still less than 50 minutes.

Custom beads are pluriBeads that fulfill particular customer needs. For example, we may indentify a potential catcher antibody (commercial or unknown), produce your individual antibody-coated pluriBeads and prepare a customized kit for you.

Universal beads are pluriBeads that are not coupled with a specific antibody yet, but allow for the binding of your own favored antibody to our catcher particles. The thus designed individual beads will bind and isolate the required targets from all kinds of biological samples (whole blood, buffy coat, tissue). They can be labeled in a batch and used when needed.

Yes, there are two different possibilities:

1. Simultaneous separation of 2 targets from one sample (at the same time): pluriBead Cascade
2. Sequential separation of more than 5 targets from 1 sample (one after the other): pluriBead Step-by-Step

Both applications generate only minimal sample loss so that they are optimal for small or precious samples. They do not require equipment apart from that provided by the kits. Main application areas are clinical studies and cell/blood banking.

A separation consists pluriBead suspension, a pluriStrainer and a Connector Ring (with Luer-Lock). For samples larger than 4ml an additional funnel should complement the separation device. The individual components are delivered with the Reagent Kit and can also be ordered separately in our shop.

The different sample materials require divergent treatment. For example, tissue requires more strainers because tissue usually requires pre-filtering. Similarly, buffy coat requires a special stabilization buffer.

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.

pluriBeads are polystyrene particles that have undergone specific surface modification. Thus, they are biocompatible for cells and show a good mass density which is important for effective incubation with the sample. pluriBeads are stable with phenol or other chemicals.

Targets are cells, proteins or microorganism from whole blood, buffy-coats, cell culture, primary cell isolation or cord blood samples. All targets can be distinguished and isolated by means of specific surface molecules (proteins, carbohydrate chains, ect.). pluriSelect’s proprietary isolation beads are surface coated with a target-specific antibody that will bind to the target and separate the target from unspecific cells or proteins. Bound targets are subsequently isolated through pluriSelect’s proprietary technology. Your targets are now ready for the use in cell culture assays, nucleic acid isolation assays or protein analysis assays.

Yes. Since pluriBead is a non magnetic isolation method, targets as well as the flow-through are not marked in any way. Thus, after separating your targets with pluriBead you can use any magnetic system for a further isolation or depletion to enrich or deplete a sub-population, e.g. you can isolate CD14 (monocytes) from CD4 (T-cells), or CD25 (Treg cells) from CD4 (T-cell), etc.

The separations beads developed by pluriSelect are not magnetic! The isolation technology is based on the separation of beads through the use of a proprietary sieving technology. That allows the application directly in whole blood, buffy coat or other sample material without Ficoll gradient or similar preparation steps. Furthermore, pluriBeads cannot be phagocytized by granulocytes.

After cell isolation the pluriBeads can be detached from the cells easily and completely. Also, pluriBeads can be used with larger sample volumes, e.g. 45ml whole blood or 45ml buffy coat.

The pluriBead technology, is based on the isolation of specific cells with the help of plastic (polystyrene) beads coated with specific antibodies. Target cells bind to the surface of these beads and can afterwards be separated from the sample (e.g. whole blood, buffy coat) by using a pluriStrainer. There is no need for sample preparation (e.g. density gradient) or centrifugation.

pluriBead can be used at room temperature and even at 37°C.

Cell separation with pluriBead is very fast: cells for RNA and protein analysis can be gained in less than 15 min, for cell culture purposes in less than 45 min. 

You can isolate more than 5 targets from one sample. The technology can also be combined with other available systems, such as magnetic cell isolation technology.

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.

The different sample materials require divergent treatment. For example, tissue requires more strainers because tissue usually requires pre-filtering. Similarly, buffy coat requires a special stabilization buffer.

That is very simple. Just add 50 µl pluriSpin solution per 1 ml whole blood. In case you would like to use buffy coat, you have to calculate with the number of concentration, e.g. 50 ml buffy coat from a 500 ml whole blood bag is a 10x concentration = 500 µl pluriSpin per 1 ml buffy coat.

Since pluriSpin is used for a negative cell separation no beads will be attached to the target cells.

pluriSpin beads are polystyrene particles that have undergone specific surface modification. Thus, they are biocompatible for cells and show a good mass density which is important for effective incubation with the sample.

Yes. Since pluriSpin is a non magnetic negative isolation method, the untouched target cells are not labeled with beads or antibodies in any way. Thus, after separating your cells with pluriSpin you can use any magnetic system for a further isolation or depletion to enrich or deplete a sub-population, e.g. you can isolate CD25 (Treg cells) from CD4 (T-cell).

The pluriSpin separations beads developed by pluriSelect are not magnetic! The isolation technology is based on the depletion of unwanted cell by the pluriSpin beads in combination with a density gradient centrifugation. It is comparable with the Rosettesep™ technology from the company of Stemcell Technologies® and can be used directly in whole blood, buffy coat, bone marrow or cord blood.

Another chance for the negative isolation is the use of magnetic beads. For this you first have to prepare PBMC via density gradient centrifugation and then you can deplete the unwanted cell populations.

The pluriSelect technology, pluriSpin, is based on the specific depletion of cells with the help of plastic (polystyrene) beads coated with antibodies in combination with a density gradient centrifugation (Leuko Spin Media, Ficoll®, Leucoprep®, Biocoll® ect). Target cells bind to the surface of the pluriSpin beads. During the density gradient centrifugation the pluriSpin beads will go through the gradient and will pull the “unwanted” cells with them. 

There is no need for sample preparation (e.g. density gradient) or centrifugation. pluriSpin can be used cooled or at room temperature. Cell separation with pluriSpin is very fast isolation method for untouched cells. You can isolate more than 5 targets from one sample. The technology can be combined with other available systems, such as magnetic cell isolation technology. pluriSpin Beads and density gradient media is all you need for the cell separation. Thus, no extra tools are needed and no extra costs arise.

With our pluriStrainers, we offer two different materials for a selection of mesh sizes - PET and nylon.

To help you decide which material is most suitable for your application, we have summarised the most important properties of the PET and nylon mesh below:

Both materials are suitable without restrictions if you are working with aqueous solutions or media in the physiological pH range (e.g. wash buffer, cell culture medium, PBS, HBSS, RBC lysis buffer).

If the cells, spheroids, tissue or other sample material are to be mechanically disrupted before flow through the pluriStrainer, the PET mesh should be chosen because of its higher strength and stability.

If you are planning a phenol-chloroform extraction on the pluriStrainer, we recommend the PET mesh (an incubation period of 15min showed no limitations for the quality of the DNA, RNA or proteins in our tests).

If you use acetone, the nylon mesh is recommended because of its better resistance.

Both materials have suitable stability against ethanol.

If you are working with more concentrated acids or bases, you should choose PET mesh for acids and nylon mesh for basic solutions.

Please note:

The information on stability is a guideline and does not exclude individual application. Material stability also depends on the duration of contact. A short contact with a chemical may have no effect even on a more sensitive mesh material, so that both PET and nylon can be considered for your application.

For the PET mesh, you have a wider range of mesh sizes available. Therefore, if the pore size of the pluriStrainer mesh is the relevant factor for you, you will usually find the right one for you in our pluriStrainers with PET mesh!

	PET		Nylon
Aqueous liquids in physiological pH ranges (e.g. wash buffer, cell culture media, PBS, HBSS, RBC Lysis Buffer)	✔	No restrictions known	✔	No restrictions known
Phenol-Chloroform (e.g. for DNA extraction on the strainer)	✔	Limited resistance but suitable for short-term contact (Tested for DNA, RNA and protein extraction for 15 min incubation time with proper results)	~	Limited resistance Not tested
Acetone resistance	~	Limited resistance Short-term contact is possible if necessary	✔	Good
Ethanol resistance	✔	Good	✔	Good
Acid resistance	✔	Good for most of solutions used in laboratory	~	Limited more restrictions on the solutions used in the laboratory
Alcaline resistance	~	Limited more restrictions on the solutions used in the laboratory	✔	Good for most of solutions used in laboratory
Mechanical disruption (e.g. grinding of cells, cell cluster, spheroids, tissue, plant material)	✔	Good Robust, stiff	~	Limited Soft, flexible
Maximum working temperature dry		150°C		115°C