B cell malignancies research

  • Isolation and analysis of B-CLL cells 
  • Isolation and analysis of ROR1-expressing B cells 

Application data for research on B cell malignancies by workflow step

B cell separation

Magnetic B cell separation with MACS® Technology

Whether you require highly purified populations separated by positive selection or untouched cells from whole blood, MACS Technology has a solution for you.

Isolation of ROR1+ B cells 

For the isolation of ROR1+ B cells, we have developed the Anti-ROR1 MicroBead Kit, human. Using this kit, ROR1+ cells are indirectly magnetically labeled with an Anti-ROR1-PE antibody and Anti-PE MicroBeads. The isolated ROR1+ B cells are then ready for any downstream analysis. ROR1+ cells can be enriched to high purities and numbers suitable for further research processing – even from samples with very low ROR1+ B cell frequencies, e.g., in minimal residual disease (MRD).

Enrichment of ROR1+ cells
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Enrichment of ROR1+ cells

Enrichment of ROR1+ cells from PBMCs of a donor with MRD. ROR1+ cells were isolated using the Anti-ROR1 MicroBead Kit, either manually. Cells were fluorescently labeled with Anti-ROR1-PE and CD19-APC and analyzed by flow cytometry. Purities of the manually and automatically isolated cells amounted to 94% and 98%, and yields amounted to 45% and 97%, respectively.

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Application note: 
Sensitive analysis and isolation of ROR1+ B cells for research on chronic lymphocytic leukemia 

Isolation of CLL B cells directly from blood products using MACSxpress® Technology

MACSxpress® Technology is designed to isolate viable and functional B cells fast and conveniently from blood and blood products. This cell isolation technology is based on micro-sized MACSxpress Beads, which allow the removal of non-target cells by immunomagnetic depletion using the MACSxpress Separator, a powerful permanent magnet. All without the need of density gradient centrifugation or red blood cell lysis. 

MACSxpress® Whole Blood B-CLL Cell Isolation Kit, human

Untouched B-CLL cells were isolated from 30 mL of human EDTA-anticoagulated whole blood using the MACSxpress® Whole Blood B-CLL Cell Isolation Kit, a MACSmix™ Tube Rotator, and a MACSxpress Separator. The isolated cells were fluorescently stained with CD19-PE and CD45-VioBlue® and analyzed by flow cytometry using the MACSQuant® Analyzer 10. Cell debris, non-leukocytes, and dead cells were excluded from the analysis based on CD45 expression, scatter signals, and propidium iodide fluorescence. This way a high cell purity can reached in a very short time.

Before separation
After separation
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Scientific poster: 
Isolation of functional cells straight from buffy coat, LRSC, and Leukopak® 

Maike Schlemminger, Silke Weber-Lohmann, Martin Kolnik*, Zhong Yu, Susanne Höher-Peters, and Gregor Winkels 
 

Multiparameter flow analysis of malignant B cells

As the final step in this workflow, flow analysis plays an important part. The following flow reagents can help you make sure that the data you generate on your flow cytometer are reliable and consistent: 

  • REAfinity™ Recombinant Antibodies – recombinantly generated antibodies that ensure lot-to-lot consistency and eliminate background signal
  • MACSplex™ Cytokine Kits – analysis of multiple soluble cytokines in one sample using any standard flow cytometer
  • Cytokine Secretion Assay – Detection Kits – flow analysis of cytokine secretion at a detection level of one in a million
  • Build your own B cell flow panel with our Flow Panel Builder
Flow cytometric analysis of ROR1+ cells
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Flow cytometric analysis of ROR1+ cells and other immune cells in PBMCs from a CLL sample

Identification of ROR1+ cells and other immune cells in PBMCs from a CLL sample. Cells were labeled with CD20-VioBlue, CD43-FITC, CD5-PE, CD79b-PerCP-Vio700, CD19-PE-Vio770, Anti-ROR1APC, and CD3-APC-Vio770, and analyzed using the MACSQuant Analyzer 10. CD19+ROR1+ B cells are shown in magenta, CD19+ROR1 B cells in light blue, T cells in yellow, and NK cells in black.

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Application note: 
Sensitive analysis and isolation of ROR1+ B cells for research on chronic lymphocytic leukemia 

Advanced analysis of B cell malignancies with Express Modes

Reproducibility is a key factor in flow cytometry experiments. It can be influenced by biological material, reagents and instrumentation, as well as analysis of acquired data, whereby the operator leads to variable results. 
Our MACSQuantify™ Express Modes are software add-ons for our MACSQuant® Analyzer Flow Cytometers. They are algorithm-based gating tools that allow for an unbiased and user-independent analysis, resulting in improved reproducibility of flow cytometry data and statistics. Based on real data, analysis and acquisition, scripts facilitate automated, application-specific gating on the actual population distribution. 

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Express mode on the MACSQuant Analyzer

After performing cell separations it is vital to determine the purity and yield of target cell populations by flow cytometry. With the MACS Control Cocktails, it is simple to qualify the separation efficiency

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