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Cell subsets contained in tumor tissue
| Cell subset | Frequency in tumor | Markers | Function |
| Tumor cells | As little as <10% | Need to be determined empiricially | Tumor growth |
| TILs | 2–50% | CD45 and additional subtype markers | Depending on the TIL subtype: tumor suppression or tumor promotion |
| Fibroblasts | Can cover >50% of the area in tissue sections, but frequency is lower |
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| Endothelial cells and pericytes | <4% |
| Tumor vascularization |
MACS Handbook:
Tumor tissue (mouse)
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Cancer research solutions (brochure)
To assure oxygen and nutrient supply, tumors induce vascularization by endothelial cells and pericytes. However, the frequency of these cell types usually is below 4%. Endothelial cells can be detected using the markers CD31 and CD146. Specific detection also requires marker combinations such as CD45–CD31+.
In general, the frequency of fibroblasts and endothelial cells is lower in mouse tumor models compared to the corresponding human tumors due to the faster tumor growth kinetics in mice.
Miltenyi Biotec has developed dedicated applications to isolate and characterize different cell types from tumor tissue.
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MACS Handbook:
Magnetic cell separation
At a glance: Kits and reagents for the separation of tumor cells
| Starting material | Isolation strategy | Comments | Automation possible | Product |
| Single-cell suspension from tumor tissue | Isolation by depletion of non-target cells | No specific tumor markers are required to isolate tumor cells | Yes | Tumor Cell Isolation Kit, mouse |
| Single-cell suspension from tumor tissue | Positive selection of target cells | Isolation of EpCAM+ tumor cells | Yes | CD326 (EpCAM) MicroBeads, mouse |
| Single-cell suspension from tumor tissue | Isolation by depletion of non-target cells | Depletion of TILs | Yes | CD45 MicroBeads, mouse |
| Single-cell suspension from xenograft tissue | Isolation by depletion of non-target cells | Isolation of human cells from xenografts | Yes | Mouse Cell Depletion Kit |
Solid tumors are infiltrated by cells of non-tumor origin, including heterogeneous lymphocyte subpopulations, fibroblasts, and endothelial cells. The amount and composition of infiltrating cells is highly variable, which makes the analysis of primary tumor samples difficult. In addition, the culture of mouse tumor cells is frequently hampered by fibroblasts overgrowing the target cells, which can bias assays such as drug sensitivity tests.
To overcome these limitations, Miltenyi Biotec has developed the Tumor Cell Isolation Kit, mouse, which provides a fast and easy method to isolate untouched mouse tumor cells from dissociated primary tissue. This cell isolation procedure is based on the comprehensive depletion of cells of non-tumor origin such as TILs, fibroblasts, and endothelial cells based on MACS® Technology. The underlying negative selection strategy enables the isolation of tumor cell populations without knowledge of surface protein expression on these cells. Even from tissues that initially contain only low numbers of tumor cells (<20%), purities of greater than 95% can be achieved in less than 20 minutes. Enriched tumor cells remain untouched, improving any downstream analysis.
Mouse breast carcinoma cells
Isolation of mouse breast carcinoma cells. Tumor cells were purified from a dissociated mouse 4T1 tumor using the Tumor Cell Isolation Kit, mouse, an LS Column, and a QuadroMACS™ Separator. The 4T1 tumor cells were GFP-labeled prior to tumor induction. The unseparated fraction and the negative fraction were analyzed for GFP and lineage markers by flow cytometry using the MACSQuant® Analyzer.
Mouse colon carcinoma cells
Isolation of mouse colon carcinoma cells. GFP-expressing CT26.WT were isolated from a heterogeneous cell population using the Tumor Cell Isolation Kit, mouse. Subsequently, the unseparated fraction (A) and the mouse tumor cell fraction (B) were cultured for three days and stained with a CD90.2 antibody (red). Cell nuclei were stained with DAPI (blue). Tumor cells were detected by their GFP expression (green).
Isolation of tumor cells from tumors induced by the 4T1 cell line. Tumors were dissociated using the Tumor Dissociation Kit, mouse and the gentleMACS Octo Dissociator. CD45+ cells were depleted using CD45 MicroBeads, a MidiMACS™ Separator, and an LS Column. CD326 (EpCAM)+ cells were isolated using CD326 (EpCAM) MicroBeads, a MidiMACS Separator, and an LS Column. The cells were fluorescently stained with CD45-PE and CD326 (EpCAM)-APC and analyzed by flow cytometry using the MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
Related PDFs:
MACS Handbook:
Tumor cells (human)
At a glance: Kits and reagents for the separation of TILs from tumor tissue
| Starting material | Isolation strategy | Comments | Automation possible | Product |
| Single-cell suspension from tumor tissue | Positive selection of target cells | Isolation of all TILs | Yes | CD45 (TIL) MicroBeads, mouse |
| Single-cell suspension from tumor tissue | Positive selection of target cells | Isolation of CD4+ TILs | Yes | CD4 (TIL) MicroBeads, mouse |
| Single-cell suspension from tumor tissue | Positive selection of target cells | Isolation of CD8+ TILs | Yes | CD8 (TIL) MicroBeads, mouse |
| Single-cell suspension from tumor tissue | Positive selection of target cells | Isolation of CD4+ and CD8+ TILs | Yes | CD4/CD8 (TIL) MicroBeads, mouse |
Immunotherapy against cancer has proven clinical efficacy and tremendous potential in multiple tumor entities. Syngeneic mouse tumor models represent excellent systems to analyze effects of immunotherapy as they possess a fully competent immune repertoire. However, the amount and composition of tumor-infiltrating leukocytes (TILs) is highly variable, complicating the targeted analysis of subpopulations. In particular, small subpopulations might escape analysis as they might be lost in the background noise. Immunophenotyping of TILs by flow cytometry is time consuming and data processing highly work intensive. Therefore, efficient pre-enrichment of TILs is desirable to increase sensitivity of analysis and save time and effort during flow cytometry. Miltenyi Biotec has established an automated workflow combining gentle tissue dissociation with specific isolation of TILs. The process preserves cell surface epitopes and thus overcomes bias in immunophenotyping, which is a frequent issue with other tissue dissociation procedures that rely on aggressive or impure enzymes. A list of preserved epitopes can be downloaded from the Related Resources panel to the right.
CD45 (TIL) MicroBeads, mouse enable the positive selection of the overall CD45+ TIL population from suspensions derived from solid mouse tumors, e.g., CT26.WT, 4T1, and B16F10.
Isolation of TILs from tumors induced by the B16F10 cell line. A tumor induced by the B16F10 cell line was dissociated using the gentleMACS™ Octo Dissociator with Heaters in combination with the Tumor Dissociation Kit, mouse. CD45+ TILs were isolated from the resulting single-cell suspension using CD45 (TIL) MicroBeads, mouse, an MS Column, and a MiniMACS™ Separator.
Cells were fluorescently stained with CD45-PE and Labeling Check Reagent-VioBlue® and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
A scientific poster presenting TIL isolation from syngeneic mouse tumors with CD45 (TIL) MicroBeads can be downloaded from the Related Resources panel to the right.
CD4/CD8 (TIL) MicroBeads are optimized for the isolation of CD4+ and CD8+ TILs from single-cell suspensions of mouse tumors. For the specific isolation of individual CD4+ and CD8+ TIL subpopulations CD4 and CD8 (TIL) MicroBeads are available.
Isolation of CD4+ and CD8+ TILs from a tumor induced by the B16-OVA cell line. The tumor was dissociated using the gentleMACS™ Octo Dissociator with Heaters in combination with the Tumor Dissociation Kit, mouse. CD4+ and CD8+ TILs were isolated from the single-cell suspension using CD4/8 (TIL) MicroBeads, two MS Columns, and a MiniMACS™ Separator. Cells were fluorescently stained with CD4-PE-Vio® 615 and CD8b-VioBlue® and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and Viobility™ 405/520 Fixable Dye fluorescence.
Related PDFs:
Epitope preservation list (mouse)
Analysis of tumor-infiltrating T cells made easy – Enrichment greatly improves sensitivity of flow cytometry analysis (scientific poster)
Optimized workflow improves the characterization of tumor-infiltrating T cells (scientific poster)
Workflow automation and parallelization improves the isolation and analysis of tumor-infiltrating immune cell subpopulations (scientific poster)
Cancer research solutions (brochure)
At a glance: Kits and Reagents for the separation of endothelial cells
| Starting material | Isolation strategy | Comments | Automation possible | Product |
| Single-cell suspension from tumor tissue | Positive selection of target cells | Yes | CD31 MicroBeads, mouse | |
| Single-cell suspension from tumor tissue | Isolation by depletion of non-target cells | Depletion of TILs | Yes | CD45 MicroBeads, mouse |
| Single-cell suspension from tumor tissue | Positive selection of target cells | Yes | CD146 (LSEC) MicroBeads, mouse |
The frequency of endothelial cells and pericytes in tumor tissue is usually below 4%. Therefore, pre-enrichment is necessary for most downstream assays. Depletion of CD45+ cells prior to endothelial cell enrichment with CD31 or CD146 (LSEC) MicroBeads allows specific detection based on markers such as CD31 and CD146.
Flow cytometry is a suitable technique for the identification and quantification of the various lineages found in mouse tumor tissue. Expression of cell surface markers on tumor cells is highly dependent on the cells’ origin. Common epithelial markers are not available, making an analysis difficult. TILs on the other hand can be easily detected by their ubiquitous and specific expression of CD45. A multitude of TIL subpopulations can be defined by the expression of surface marker combinations. Fibroblasts express CD44, CD90.1 or CD90.2 (depending on the mouse strain), and vimentin. As these markers are also expressed by other lineages within the tumor, marker combinations are required for clear identification of fibroblasts. Endothelial cells can be detected based on the markers CD31 and CD146. However, specific detection requires marker combinations such as CD45–CD31+.
Suitable flow cytometry antibodies for immunophenotyping of tumors or the characterization of T cells from the tumor microenvironment are listed below.
Panels of REAfinityTM Antibodies for the analysis of tumor-infiltrating CD8+ T cells
| Panel 1 | Clone | Fluorochrome | Panel 2 | Clone | Fluorochrome |
| CD8ß | REA793 | VioBlue | CD8ß | REA793 | VioBlue |
| Viobility | 405/520 | Viobility | 405/520 | ||
| Lag-3 | REA776 | SVio515 | CD103 | REA789 | SVio515 |
| TIM-3 | REA602 | PE | CXCR3 | REA724 | PE |
| CD4 | REA604 | PE-Vio615 | CD4 | REA604 | PE-Vio615 |
| PD-1 | REA802 | APC | CD39 | REA870 | APC |
| CD44 | REA664 | APC-Vio770 | CD44 | REA664 | APC-Vio770 |
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