Tumor infiltrating B cells

Application notes and a scientific poster for tumor infiltrating B cells

• Isolation of ovarian cancer cells from primary human tumor samples using the gentleMACS™ Dissociator (application note)
•  Isolation of TILs after tumor dissociation significantly improves the sensitivity of single-cell immune profiling (application note)
• Increased sensitivity of flow cytometry for detection of minimal residual disease and circulating tumor cells in B Non-Hodgkin Lymphoma (application note)
• Complete workflows allow comprehensive tumor microenvironment analysis of cell subsets from limited tumor patient samples (scientific poster)

Tumor dissociation
 

In order to study tumor infiltrating B cells (TIBs), the tumor first has to be dissociated into a single cell suspension. For this, the gentleMACS™ Dissociator in combination with the Tumor Dissociation Kit, human ensures rapid and standardized dissociation of tissue samples into single-cell suspensions. Whatever the tumor entity, gentleMACS Technology provides excellent cell viability and functionality for further downstream cell separation, analysis, and culture.

Get more information on our solutions for analysis of tumor composition and microenvironment here.

Tumor infiltrating B cell separation
 

To isolate tumor infiltration B cells (TIBs) positive selection positive selection is recommended, due to the highly diverse sample composition, tumor type, and origin, as well as patient differences. Whether you require for your experiments, label free B cell or classical B cell isolation using CD19 MicroBeads or cells isolated using a flow cytometer, we have a solution for you.

The amount and composition of tumor-infiltrating leukocytes (TILs) is highly variable, complicating the analysis of B cell populations. However, TIB numbers are often very low and may escape analysis when they are lost in background noise. This can be especially challenging when performing single-cell analysis. Moreover, a prerequisite for single-cell analysis is a debris-free single-cell suspensions with a viability above 80%. When working with large cohort sizes, immunophenotyping of TIBs by flow cytometry is time consuming and the data processing can be intense. Therefore, pre-enrichment is highly desirable to increase the sensitivity of analysis, saving time and effort during flow cytometry.

REAlease® Technology allows for magnetic cell isolation with subsequent removal of any beads and labels from your cells of interest. Clever utilization of recombinant antibody fragments ensures cell surface labeling in just a few steps.  
The technology relies on recombinantly engineered antibody fragments for cell surface labeling for highly reproducible results. The REAlease Biotin Complex binds to the target cells. Labeling of the REAlease Biotin Complex with Anti-Biotin MicroBeads allows for magnetic isolation of these cells. Following cell separation, both MicroBeads and REAlease Biotin Complex are gently removed, leaving the cells bead- and label-free. Enriched cells are then suitable for magnetic re-labeling and any application where label-free cells are essential. 

The MACSQuant® Tyto® Cell Sorter is revolutionizing cell sorting. Our patented microchip-based technology opens up new possibilities with high-speed multiparameter cell sorting in the safety of a fully enclosed and sterile cartridge system, the MACSQuant Tyto Cartridge. This enables gentle sorting of TIBs, leading to high target cell purity and viability of theses rare cells.

B cell culture 

When working with rare cell populations such as TIBs ex vivo cultivation is often used to increase B cells in numbers; either to study their biology, or transform them and analyze their antibody production as well as specificity. This can often be difficult, however using the B Cell Expansion Kit, human, streamlines and simplifies the process. The formulation offers the perfect solution to expand and activate human B cells from various sources which are fully functional and ready for any downstream application.

Reliable activation and expansion of B cells is a clear requirement for effective downstream experiments. The B Cell Expansion Kit consist of a recombinant CD40L multimer, a cross-linking antibody, and IL-4. Cross-linking of CD40 expressed on B cells leads to cell activation and proliferation. Combining CD40L together with IL-4, which is a known growth factor for activated B cells, has been shown to be crucial for effective activation and proliferation of B cells.

Expanded B cells display an activated phenotype with a distribution of memory and plasma cells similar to the original fraction. The amounts of naive B cells decreased under all culture conditions on day 14. Likewise, reports describe that naive B cells have a shorter survival rate. 
 

Multiparameter flow analysis of B cells

As the final step in many workflows, flow analysis plays an important part in the analysis of TIBs. Definition and distribution of subsets, differentiation and activation status of the cell in the tumor are of special interest. Our flow reagents ensure that the data you generate on your flow cytometer is reliable and consistent:

• REAfinity™ Recombinant Antibodies
  Recombinantly generated antibodies that ensure lot-to-lot consistency and eliminate background signal
• Build your own B cell flow panel with our Flow Panel Builder
• 8-Color Immunophenotyping Kit, human
  The 8-Color Immunophenotyping Kit simplifies the flow-cytometric evaluation of cell fractions for immunofluorescent staining of single-cell suspensions from human tissue or many other starting materials. As well as human B cells, the kit cocktail has been designed for reliable identification of human monocytes, neutrophils, eosinophils, and T, B, and NK lymphocyte populations as well as CD4⁺, CD8⁺, and CD56⁺ CD3⁺ T cell subsets.