Isolation and characterization of tumor infiltrating leukocytes (TILs)

  • Tumor dissociation with preserved cell surface epitopes
  • TILs pre-enrichment for improved down stream analysis
  • From manual single sample separation up to fully automated high throughput separation
     

Application protocol for enrichment of TILs from mouse tumor tissue

Protocol 
Enrichment of tumor-infiltrating lymphocytes (TILs) from mouse tumor tissue
 

MACS Handbook:

Tumor cells (human)

Tumor cells (mouse)

Tumor tissue (human)

Tumor tissue (mouse)

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Application data by workflow step

Tumor dissociation and storage

Comparison between MACS® Tissue Storage Solution and competitor products
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Optimized storage for up to 48 hours

Freshly dissected tumor tissue can be stored for up to 48 h without compromising cell viability or causing unwanted effects like cell activation or apoptosis using MACS® Tissue Storage Solution.

IFN-y release of TILs generated with the gentleMACS™ Dissociator compared to overnight digestion
 
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Preserved anti-tumor functionality of TILs

Secretion of IFN-γ is one of the hallmark functions of antigen-specific T cells encountering the respective tumor antigen. Combined mechanical/enzymatic processing of TILs with the gentleMACS™ Dissociator is gentle to cells and results in profound secretion of IFN-γ upon tumor cell coculture.

Recovery of CD8+ TILs from B16-F10 tumors dissociated in presence or absence of TDK enzymes
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Enzymatic tumor dissociation for efficient recovery of TILs

Standardized processing of tumor samples using the Tumor Dissociation Kit (TDK) together with the gentleMACS™ Dissociator enhances reproducibility and was essential for recovery of key populations of TILs.

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Magnetic isolation of TIL populations

The amount and composition of tumor-infiltrating leukocytes (TILs) is highly variable, complicating the analysis of individual subpopulations. When working with large cohort sizes, even immunophenotyping of TILs by flow cytometry is time consuming and data processing highly work intensive. Therefore, pre-enrichment of TILs is highly desirable to increase the sensitivity of analysis and save time and effort during flow cytometry. 
Isolation and analysis of CD4+,CD8+and CD4+/CD8+ T cells
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Isolation of CD4+, CD8+, and pan T cells from syngeneic mouse tumors

Using CD4+, CD8+, and pan T cell specific MACS MicroBeads for magnetic cell isolation (MACS® Technology) directly from dissociated tumor tissue delivers high purities of isolated TILs above 80%. 

This pre-enrichment of TILs greatly reduces time and costs of downstream analysis while the quality of data analysis significantly increased.


Isolation and analysis of CD45+ T cells
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Isolation of CD45+ T cells from syngeneic mouse tumor

Isolation of TILs directly from dissociated tumor tissue can be improved by using the CD45 (TIL) MicroBeads for magnetic cell sorting. 

Pre-enrichment of TILs significantly improves subset analysis and does not affect the composition of infiltrating immune cell populations.

Isolation of CD45+ T cells step-by-step

These short video protocols show the quick isolation of CD45+ TILs – either manual for less samples or fully automated for high-throughput cell isolation approaches.

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Video
Magnetic separation of TILs with LS Columns

Watch how easy it is to separate tumor-infiltrating lymphocytes using MACS Technology.

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Video
High-throughput magnetic separation of TILs with the MultiMACS™ Cell24 Separator

Watch how the MultiMACS Cell24 Separator Plus and the MultiMACS X can enhance your workflow of isolating CD45-positive tumor-infiltrating leukocytes.

MACS Handbook:

Magnetic cell separation

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Analysis of TILs
 

Phenotypic characterization of TILs by flow cytomety can be performed using optimized panels of fluorochromes-conjugated REAfinity™ Recombinant Antibodies. In addition, the scientific poster below gives a comprehensive overview on how to optimize single workflow steps for improved TILs analysis.
Panel 1
CloneFluorochromePanel 2
CloneFluorochrome
CD8βREA793VioBlue®CD8βREA793

VioBlue

Viobility405/520Viobility405/520
Lag-3REA776VioBright™ 515
CD103REA789VioBright 515 
TIM-3REA602PECXCR3REA724PE
CD4REA604PE-Vio® 615CD4REA604PE-Vio 615
PD-1REA802APCCD39REA870APC
CD44REA664APC-Vio 770CD44REA664APC-Vio 770
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Seminar
Isolation and analysis of tumor-infiltrating immune cell populations

Listen to the lecture delivered by Dr. Cesar Evaristo during Tri-Con 2017 at the Moscone North Conference Center, San Francisco, CA, and learn more about complete workflows for isolation and analysis of immune cells.

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Scientific poster
Optimized workflow improves the characterization of tumor-infiltrating T cells

César Evaristo, Ramona Siemer, David Agorku, Janina Brauner, Olaf Hardt, Christian Dose, and Anne Richter

Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany


 

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