MACS Handbook

Cells in tumor tissue

1 Introduction

Solid tumors are a mixture of many different cell types from various lineages. Differentiation and quantification of the various lineages and cell subsets found in tumor tissue can be done using combinations of origin-dependent cell surface and intracellular markers.
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Tumor tissue (human)

2 Cell subsets, frequencies, and marker expression

At a glance: Cell types in tumor tissue
Cell type or subsetFrequency in tumorMarkersFunctions
Tumor cells (in carcinomas)EpCAM, E-cadherin. Intracellular markers, such as lineage-specific cytokeratin subtypes, can also be used.Tumor growth
Cancer stem cells (CSC)Highly variable (reports of 0.0001–40%)CD133, CD44, CD24, EpCAM, ABCB5, CD200; combinations are most effective for detection
  • Possess self-renewal and differentiation capacity
  • Involved in tumor growth and metastasis
Tumor-infiltrating lymphocytes (TIL)2–50 %CD45; plus subpopulation-specific surface marker combinationsDifferent immune subpopulations exhibit either tumor-suppressive or tumor-promoting functions.
FibroblastsCan cover >50 % of area inside and around tumor sections, but frequency is typically low.CD44, CD90, vimentin; combinations are usually required, such as EpCAM/CD45/CD90+
  • Modulate tumor extracellular matrix
  • Can transform the tissue into dense fibrotic structures (desmoplasia)
  • Active role in tumor growth promotion and metastasis
Endothelial cells and pericytes<4 %A broad range including CD31, CD144, CD34, CD146. Specific detection requires combinations like CD45–/CD31+Tumor vascularization
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2.1 Tumor cells

Tumor cells inside a solid tumor are often outnumbered by stromal cells in many tumor entities. One prominent example is pancreatic cancer where frequently less than 10% (internal data) of all cells inside the lesion are tumor cells (PMID: 14625861). The expression of cell surface markers on tumor cells is highly dependent of their origin. Epithelial markers such as EpCAM or E-cadherin are most commonly used to detect tumor cells. In addition, intracellular markers, such as lineage specific cytokeratin subtypes, can also be used.

2.2 Cancer stem cells (CSCs)

The existence of cancer stem cells has been debated, yet this cell type is intensely studied and has been reported in several solid tumors, including brain, breast, colon, ovary, pancreas, prostate, skin cancers and multiple myeloma. As cells with the potential to self-renew and give rise to all cell types found in a tumor, CSCs are implicated in relapse and metastasis, and are interesting targets for cancer therapies. Studies have reported a broad range of CSC frequencies in tumors, which has led to discussion about possible phenotypic switching and the impact of experimental methods on CSC function (PMID: 27121191, 23950884).

2.3 Tumor infiltrating lymphocytes or leukocytes (TILs)

The most complex lineage of non-malignant cells infiltrating a tumor are immune cells. All immune subpopulations can be found in a tumor and each displays either tumor-suppressive or tumor-promoting functions. Cytotoxic T cells, T helper cells, and M1 macrophages are tumor-suppressive, whereas regulatory T cells or M2 macrophages usually promote tumor growth (PMID: 24122236). The frequency of TILs is highly variable and ranges mostly between 2% and 50%. The overall TIL population can easily be detected by their ubiquitous and specific expression of CD45, and dozens of TIL subpopulations can be defined by the expression of surface marker combinations.

2.4 Fibroblasts

Cancer-associated fibroblasts play a key role in modulating a tumor’s extracellular matrix, and can transform tissue into dense fibrotic structures (desmoplasia), a common phenomenon in many carcinomas, including breast and pancreatic cancer (PMID: 14581350). Fibroblasts also play an active role in tumor growth promotion and metastasis; for example, they secrete cytokines and pro-tumorigenic extracellular matrix molecules (PMID: 19308067). Tumor tissue sections can include fibroblasts inside the tumor and in surrounding tissue, at times covering over 50% of the section area due to their large size and deposition of extracellular matrix that fills a significant portion of tumor space. Their actual frequency, however, is quite low. Fibroblasts express CD44, CD90, and vimentin. As these markers are also expressed by other lineages inside the tumor, combinations like EpCAM/CD45/CD90+ are used to identify fibroblasts.

2.5 Endothelial cells and pericytes

To ensure oxygen and nutrient supply, tumors induce vascularization, which includes buildup of endothelial cells and pericytes. However, the frequency of these cell types is usually below 4%. Endothelial cells can be detected using a broad range of markers including CD31, CD144, CD34 and CD146. However, specific detection also requires marker combinations such as CD45/CD31+.

3 Miltenyi Applications for cells from tumor tissue

Miltenyi Biotec has created dedicated applications to isolate and characterize different cell types from tumor tissue.

4 Sample Preparation of tumor tissue

Tumor tissues must be dissociated into a single-cell suspension for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis. For tumor tissue, this may require use of digestion enzymes. For details, see chapter Human cells and organs – Tumor tissue

5 Magnetic separation of cells and exosomes from tumor tissue

Miltenyi Biotec has developed numerous products for the magnetic separation of the various cell types that can be found in tumor tissue. For details on MACS Cell Separation Technology,  see the MACS handbook chapter Magnetic cell separation

5.1 Isolation of tumor cells from human tumors

At a glance: Kits and reagents for the separation of tumor cells from tumor tissue
Starting materialIsolation strategyCommentsAutomationProduct
Single-cell suspension from tumor tissueIsolation via depletion of non-target cellsNo specific tumor cell markers are required to isolate tumor cellsYesTumor Cell Isolation Kit, human
Single-cell suspension from tumor tissuePositive selection of target cellsIsolation of EpCAM+ tumor cells YesCD326 (EpCAM) MicroBeads, human
Single-cell suspension from melanoma tissuePositive selection of target cellsIsolation of cells expressing melanoma-associated chondroitin sulfate proteoglycan (MCSP)YesAnti-Melanoma (MCSP) MicroBeads, human
Single-cell suspension from melanoma tissuePositive selection of target cellsIsolation of MCSP+ cellsYesMelanoma Cell Enrichment and Detection 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 and patient dependent, and their presence interferes with downstream analyses. They may hybridize to probes on microarrays and significantly reduce sensitivity of next-generation sequencing or proteome analyses. Cultures of human tumor cells are frequently hampered by fibroblasts overgrowth, which can bias assays such as drug sensitivity tests.

To overcome these limitations, Miltenyi Biotec has developed the Tumor Cell Isolation Kit, human, a fast and easy method to isolate untouched human tumor cells from primary tissue. The procedure of this kit comprehensively depletes cells of non-tumor origin by combining automated tissue dissociation and magnetic cell sorting. A negative selection strategy enables isolation of the tumor cell population without knowledge of their surface protein expression. Tissues with initially low numbers of tumor cells (<20 %) can reach purities of higher than 95 % in less than 20 minutes.

Scientific posters describing the use of Miltenyi Biotec tumor cell isolation kits can be downloaded from the Related Resources panel to the right.

Tumor Cell Isolation Kit, human.
(A) Original fraction
(B) Isolated tumor cells

Tumor cell isolates of high purity achieved with the Tumor Cell Isolation Kit, human. Primary human tumor tissue after dissociation, separation and 7 days of culture. (A) Original fraction, (B) untouched tumor cells isolated using the Tumor Cell Isolation Kit, human. Cells in both images are stained for vimentin (red), EpCAM (green) and DAPI (blue).

5.2 Isolation of tumor cells from xenograft models

At a glance: Kits and reagents for the separation of tumor cells from xenograft tissue
Starting materialIsolation strategyCommentsAutomationProduct
Single cell suspension from xenograft tissueIsolation via depletion of non-target cellsIsolation of human cells from xenograftsYesMouse Cell Depletion Kit

During the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin including heterogeneous lymphocyte subpopulations, fibroblasts, and endothelial cells. The level of infiltration is highly variable and dependent on multiple factors like tumor subtype, growth rate, and region of transplantation. The contaminating mouse cells can lead cross hybridization with human probes on microarrays and mouse signals significantly reduce sensitivity of next-generation sequencing and proteome analysis. Cultures of human tumor cells are frequently hampered by murine fibroblasts efficiently plating and overgrowing the target cells.

The Mouse Cell Depletion Kit offers a fast and easy method to comprehensively deplete all cells of murine origin from xenograft tissues. Even tumors with high numbers of mouse cells (>60 %) can be cleaned to purities of human tumor cells greater than 98 % in less than 20 minutes (PMID: 26607327, 27501218). Because antibodies specific only to mouse cells are used, target cells remain “untouched” and the procedure can be used for any xenograft material, without need for a positive marker expressed on the human cells.

Mouse Cell Depletion Kit. 
(A) Original fraction
(B) Isolated human tumor cells

High purity of human tumor cell population isolated from xenograft tissue using the Mouse Cell Depletion Kit. Xenograft tissue after dissociation, separation and 3 days of culture. (A) Original fraction, (B) human tumor cells isolated using the Mouse Cell Depletion Kit. Cells in both images are stained for vimentin (red), EpCAM (green) and DAPI (blue).

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5.3 Isolation of cancer stem cells (CSC)

At a glance: Kits and reagents for the separation of CSCs from various starting materials
Starting materialIsolation strategyCommentsAutomationProduct
 Single-cell suspension from tumor tissuePositive selection of target cellsYesCD133 MicroBead Kit – Tumor Tissue, human
Single-cell suspension from tumor tissuePositive selection of target cellsYesCD90 MicroBeads, human
Single-cell suspension from tumor tissuePositive selection of target cellsYesCD44 MicroBeads, human
Single-cell suspension from tumor tissuePositive selection or depletion of target cellsUses biotin-labeled antibodies and biotin-coupled MicroBeadsYesCD24 MicroBead Kit, human
Single-cell suspension from bone marrow or cord bloodPositive selection of target cellsYesAnti-ABCG2 (CD338) MicroBeads, human
Single-cell suspension from tumor tissuePositive selection of target cellsYesAnti-SSEA-4 MicroBeads, human
Single-cell suspension from tumor tissuePositive selection of target cellsYesAnti-LGR5 MicroBeads, human

Growing knowledge concerning tumor heterogeneity has increased the importance of analyzing tumor resident and stem cell subpopulations. Cancer stem cells (CSC), or tumor initiating cells, have gained substantial interest in oncology as they have been shown to play a crucial role during tumor growth and metastasis.

Isolation and analysis of CSCs even from single tumor entities like melanoma has generated contradictory results among different research groups. Some of this variation can be explained by the different dissociation and isolation methods used. The procedure used for dissociation of primary tissue prior to staining may cause technical bias. Aggressive proteases, such as trypsin, cleave off several CSC surface markers like ABCB5 or CD44, and may cause incorrect profiles of the differential expression of sensitive markers (PMID: 21393506). Glycosylation and splice isoform-dependent epitopes, as found for CD24 or CD133, are differentially expressed among tissues and cell states, which impacts antibody-based assays. For example, the AC133 epitope but not the entire CD133 protein expression is lost upon CSC differentiation (PMID: 20068153).

Miltenyi Biotec offers a range of microbeads conjugated to monoclonal antibodies specific to the most important markers used to detect CSCs, as well as dedicated kits, such as the CD133 MicroBead Kit – Tumor Tissue, human

. This portfolio enable fast and highly reproducible CSC enumeration and isolation from various tumor tissues. Automated (and therefore user independent) procedures for dissociation of human and mouse tumor tissue without degradation of cell surface epitopes yield high amounts of single cells with approximately 90% viability. Antibody conjugates of clones that recognize epitopes relevant for CSC analysis and have been tested on cell lines and primary tumor tissue allow quantification and isolation of CSCs. For example, a sequential sorting approach enabled efficient isolation of highly viable populations of CD45-/CD24-/CD44+ breast CSCs and improved molecular characterization by next-generation sequencing based transcriptome profiling (PMID: 22771536).
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High-purity separation of human retinoblastoma cells using the CD133 MicroBeads – Tumor Tissue, human. CD133+ human retinoblastoma cells (WERI-Rb-1) were isolated from a mixture of U937 and WERI-Rb-1 cells using CD133 MicroBeads – Tumor Tissue, an MS Column, and an OctoMACS™ Separator. Cells were fluorescently stained with Labeling Check Reagent-PE and CD44-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.

5.4 Isolation of tumor-infiltrating leukocytes (TIL)

At a glance: Kits and reagents for the separation of TILs from tumor tissue
Starting materialIsolation strategyCommentsAutomationProduct 
Single cell suspension from tumor tissuePositive selection of target cellsYesCD326 (EpCAM) MicroBeads, human
Single cell suspension from tumor tissuePositive selection of target cellsYes Anti-Fibroblast MicroBeads, human 

Immunotherapy against cancer has proven clinical efficacy for multiple tumor entities. Primary human tumors are the optimal system to analyze effects of immunotherapy due to their fully competent immune-repertoire. However, amount and composition of tumor infiltrating leukocytes (TIL) is highly variable, which complicates the targeted analysis of subpopulations. Small subpopulations may be lost in background noise. Immune-phenotyping of TIL by flow cytometry is time-consuming and subsequent data processing is work-intensive, making pre-enrichment for sample debulking attractive.

Miltenyi Biotec offers an automated workflow combining tissue dissociation with TIL-specific isolation to improve and accelerate downstream analysis. Tumor dissociation is automated on the gentleMACS™ Octo Dissociator and optimized for epitope conservation to prevent the bias in immune-phenotyping caused by aggressive and impure enzymes. A list of preserved epitopes can be downloaded from the Related Resources panel to the right.

 Enrichment of untouched TILs is done by depleting non-TIL populations (debulking) using a combination of CD326 (EpCAM) MicroBeads to remove tumor cells, and Anti-Fibroblast MicroBeads to remove fibroblasts. Numerous sorting reagents specific for TIL subpopulation are also available. To learn more, refer to the chapter on the cell of interest.
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5.5 Isolation of circulating tumor cells (CTC)

At a glance: Kits and reagents for the separation of CTCs from various starting materials
Starting materialIsolation strategyCommentsAutomationProduct
Peripheral bloodPositive selection of target cellsUsed directly on whole bloodYesStraightFrom™ Whole Blood CD326 (EpCAM) MicroBeads, human
Peripheral bloodPositive selection of target cellsRed blood cell lysis requiredYesCD326 (EpCAM) MicroBeads, human
Peripheral blood or bone marrowPositive selection of target cellsNoCD326 (EpCAM) Tumor Cell Enrichment and Detection Kit, human
Peripheral bloodIsolation via depletion of non-target cellsRed blood cell lysis requiredYesTumor Cell Isolation Kit, human
Peripheral blood, bone marrow, or lymphoid tissuePositive selection of target cellsUsing cytokeratin MicroBeadsNoCarcinoma Cell Enrichment Kit, human
Peripheral blood, bone marrow, or lymphoid tissuePositive selection of target cellsUsing cytokeratin MicroBeadsNoCarcinoma Cell Enrichment and Detection Kit – Immunocytochemistry, human
Blood, bone marrow, or leukapheresis harvestPositive selection of target cellsYesAnti-ErbB-2 MicroBeads, human
Blood, bone marrow and lymphoid tissuePositive selection of target cellsNoErbB-2 Tumor Cell Enrichment and Detection Kit, human
Peripheral blood, bone marrow, or lymphoid tissuePositive selection of target cellsYesAnti-Melanoma (MCSP) MicroBeads, human
Peripheral blood, bone marrow, or lymphoid tissuePositive selection of target cellsYesMelanoma Cell Enrichment and Detection Kit
Peripheral bloodIsolation via depletion of non-target cellsIsolation of human cells from xenografts (red blood cell lysis required)YesMouse Cell Depletion Kit

Characterization of circulating tumor cells (CTCs) from blood or disseminated tumor cells (DTCs) from bone marrow is important to understanding mechanisms of cancer metastasis. Levels of CTCs in blood have been linked to patient prognosis and treatment. CTCs and DTCs are very rare, so highly sensitive methods are needed to enrich them for downstream morphological staining or molecular analysis.

 A number of Miltenyi Biotec kits and reagents have been optimized to efficiently isolate tumor cells from whole blood (StraightFrom™ Whole Blood CD326 (EpCAM) Microbeads, human) and blood products (e.g., CD326 (EpCAM) Tumor Cell Enrichment and Detection Kit, human). Each product addresses different experimental needs. For example, the Carcinoma Cell Enrichment Kit, human and the Carcinoma Cell Enrichment and Detection Kit, Immunocytochemistry, human use anti-cytokeratin MicroBeads to capture and analyze disseminated epithelial tumor cells. Similarly, the ERbB-2 Tumor Cell Enrichment and Detection Kit, human, combines a separation event for ErbB-2+ cells and then intracellular staining of cytokeratin filaments to analyze tumor cells overexpressing the human epidermal growth factor receptor by light microscopy.
StraightFrom™ Whole Blood CD326 (EpCAM) MicroBeads
Before separation
After separation

 Effective separation of tumor cells from whole blood. Whole blood samples were spiked with cells from a breast cancer cell line (SK-BR3) and tumor cells were then enriched directly from the samples using StraightFrom™ Whole Blood CD326 (EpCAM) MicroBeads and the program Posselwb on the autoMACS® Pro Separator. Cells were fluorescently stained with CD326 (EpCAM)-FITC and CD45-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.

5.6 Isolation of endothelial cells

At a glance: Kits and reagents for the separation of endothelial cells from various starting materials
Starting materialIsolation strategyCommentsAutomationProduct
Single cell suspension from tumor tissuePositive selection of target cellsYesCD31 MicroBead Kit, human
ES or iPS cell culturesPositive selection of target cellsYesCD144 (VE-Cadherin) MicroBeads, human
Single cell suspension from tumor tissuePositive selection of target cellsIdeal choice for debris-rich, low frequency samplesYesCD34 MicroBead Kit UltraPure, human
Single cell suspension from tumor tissuePositive selection of target cellsNoCD146 MicroBead Kit, human
Peripheral blood, cord blood, or leukapheresis products and EPCs from bone marrowPositive selection of target cellsEndothelial progenitor cells (EPCs) and circulating EPCs (cEPCs) NoEPC Enrichment and Enumeration Kit, human
Peripheral bloodPositive selection of target cellsFor circulating endothelial cells (cECs)NocEC Enrichment and Enumeration Kit, human

The low frequency of endothelial cells and pericytes in tumor tissue make pre-enrichment necessary for most types of downstream assays. An initial depletion of CD45+ cells prior to endothelial cell enrichment allows a more specific detection using a range of effective MicroBeads conjugated to antibodies against human CD31, CD144, CD34 and CD146. Miltenyi Biotec also offers dedicated kits like the EPC Enrichment and Enumeration Kit and the cEC Enrichment and Enumeration Kit, which include all reagents needed to enrich endothelial progenitor cells (EPCs) or circulating endothelial cells (cECs) and enumerate them based on the expression of CD34, CD133 and CD309.

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Enumeration of CD34+CD133+CD309 (VEGFR-2/KDR)+ EPCs in a pre-enriched EPC sample. Leukocytes after discrimination of debris and platelets, exclusion of dead cells and monocytes are shown. Additionally, data were gated on CD34+ cells. Cells were stained with CD309 (VEGFR-2/KDR)-APC and CD133/2 (293C3)-PE and analyzed by flow cytometry.


5.7 Isolation of fibroblasts

At a glance: Kits and reagents for the separation of fibroblasts from various starting materials
Starting materialIsolation strategyCommentsAutomationProduct
Single cell suspension from tumor tissuePositive selection of target cellsYesCD90 MicroBeads, human
Single cell suspension from tumor tissuePositive selection of target cellsYesCD44 MicroBeads, human
Single cell suspension from tumor tissuePositive selection of target cellsFibroblast-specific antigen is also known to be expressed on mesenchymal stem cellsYesAnti-Fibroblast MicroBeads, human
Single cell suspension from tumor tissuePositive selection of non-target cellsDepletion of epithelial tumor cellsYesCD326 (EpCAM) MicroBeads, human
Lymphoid tissues, tumor tissues, and nonhematopoietic tissuesPositive selection of non-target cellsDepletion of leukocytesYesCD45 MicroBeads, human

Fibroblasts express markers such as CD44, CD90, and vimentin. As these markers are also expressed by other lineages inside the tumor, combinations like EpCAM/CD45/CD90+ are useful for the isolation and detection of cancer-associated fibroblasts. MACS MicroBeads are a flexible solution to create combinations that address specific experimental needs, either as a means of depleting non-target cells like epithelial cells using the CD326 (EpCAM) MicroBeads, human, or leukocytes using CD45 MicroBeads, human, or to select specific target cells.

5.8 Isolation of exosomes

At a glance: Kits and reagents for the separation of exosomes from various starting materials
Starting materialIsolation strategyCommentsAutomationProduct
Cell culture supernatant, plasma, urine, or ascitesPositive selection of exosomesNoExosome Isolation Kit CD9, human
Cell culture supernatant, plasma, urine, or ascitesPositive selection of exosomesNoExosome Isolation Kit CD63
Cell culture supernatant, plasma, urine, or ascitesPositive selection of exosomesNoExosome Isolation Kit CD81
Cell culture supernatant, plasma, urine, or ascitesPositive selection of exosomesIsolation using all 3 major exosome markersNoExosome Isolation Kit Pan, human

Exosomes are extracellular vesicles (EVs) released by a variety of cell types either constitutively or after stimulation. EVs carry proteins, lipids, and nucleic acids like miRNAs, mRNA or even fragments of genomic DNA, and are important means for intercellular communication and transport.

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Exosomes bud off a parent cell and are engulfed by a recipient cell. Exosomes can transport a large variety of nucleic acids, lipids and proteins. Markers found on the membrane of exosomes are equal to those found on the parent cell, giving insight into the origin of an exosome.



Isolating and analyzing EVs gives insight into physiological and pathological processes within the body. Where originating cells are difficult to access, exosomes can be used to gain information about these cells. Exosomes secreted by tumor cells can be found in plasma or urine, and thus, are an important constituent of so-called liquid biopsies.

DNA extracted from plasma EVs has been used for whole genome sequencing (PMID: 26681674; 24398677). The data enable to reconstruct the mutation profile of the tumor and can be more comprehensive than a profile of a tumor biopsy. The vesicles derive from metastatic sites and therefore can represent mutations that are not present in the originating tumor.

Tumor exosomes also play important roles in tumor progression (PMID: 24766647). They can induce angiogenesis, suppress immune responses and route metastasis. In a tumor mouse model, mice pretreated with exosomes derived from a metastatic cell line exhibited metastasis even when tumor cells from a non-metastatic tumor cell line were transplanted (PMID: 26725371).

Given that the target region for metastasis can be influenced by EVs ((PMID: 26524530), tumor exosomes may support CTCs in homing to distant tissue. If EVs from tumor cells preferentially metastasizing to the lung are used to precondition mice, most metastatic sites are found in the lung. And this holds true even if the tumor cells used to inoculate the tumor are derived from a tumor that usually would mainly colonize bone marrow.

Basic research and future diagnostic applications demand reliable methods for exosome isolation. Due to their small size (ca. 100 nm), exosomes are conventionally isolated by ultracentrifugation (PMID: 24009894), which requires capital equipment and is typically performed for several hours or even overnight. High starting volumes (> 10 ml, often 50–100 ml) and low yields make isolation of exosomes from precious samples like patient plasma difficult, if not impossible. Density gradient centrifugation improves purity of isolated exosomes over classical ultracentrifugation. Precipitation of exosomes by volume-excluding reagents like PEG is fast and inexpensive, but co-precipitation of lipids or protein aggregates reduces exosome purity. Ultrafiltration or size exclusion can also be used to enrich small vesicles, though vesicles can attach to the membrane or block the filters, making yields low and unreliable.

MicroBeads enable exosome Isolation by selecting EVs according known exosome surface markers, like CD9, CD63, CD81, or all three markers concurrently (Exosome Isolation Kit Pan, human). EVs not carrying the respective markers are depleted and thus, sufficient amounts of vesicles can be isolated from as little as 0.5 to 2 mL of cell culture supernatant, urine, plasma, serum, ascites etc. The main applications for MicroBead isolation are EV analysis by downstream assays like Western Blot, MACSPlex Exosome Kit, and the analysis of exosomal RNA.

6 Characterization of tumor cell populations by flow cytometry

The expression of cell surface markers on tumor cells that can be used for flow cytometry analysis is highly dependent on their origin. Epithelial markers such as EpCAM or E-cadherin are commonly used to detect tumor cells, while the ubiquitous and specific expression of CD435 can be used to detect TILs. Fibroblasts express markers such as CD44, CD90, and vimentin. Nevertheless, lineage-specific cytokeratin subtypes, as well as combinations of surface and internal markers are needed for the specific discrimination of different cell types, subsets and subpopulations.

6.1 Flow cytometry panels

A suitable flow cytometry antibody panel for the characterization of major tumor lineages in carcinomas is CD90/EpCAM/CD31/CD45/Gly-A. A list of Miltenyi Biotec cancer-related antibodies can be downloaded from the Related Resources panel to the right.

For details about Miltenyi Biotec antibodies and dyes for cell analysis, see chapter Cell Analysis – primary antibodies.

6.2 Flow cytometry analysis of exosomes

At a glance: Kits and reagents for analysis of exosomes by flow cytometry

Starting materialCommentsProduct
Cell culture supernatant, ascites, urine, or Isolated exosomesDetection of 37 exosomal surface epitopes plus two isotype controlsMACSPlex Exosome Kit, human

The tetraspanins CD9, CD63, or CD81 are used as exosomal markers (PMID: 9685355), while surface proteins derived from the membrane of releasing cells enable inferences about cell origin (PMID: 12154376). Nevertheless, the small size of exosomes (30–100 nm) makes it difficult to analyze them directly by flow cytometry (PMID: 25688721).

Miltenyi Biotec established a multiplex bead-based assay consisting of capture and detection antibodies for flow cytometry analysis of exosome surface protein composition (PMID: 26901056). The MACSPlex Exosome Kit is an easy and fast method to analyze 39 surface proteins, including 2 negative controls, on extracellular vesicles. Each bead population bears monoclonal antibodies recognizing a surface epitope. By incubating the MACSPlex Beads with the exosome sample, vesicles are captured by the respective beads. The bound exosomes are then stained by APC-conjugated antibodies. With a diameter of about 4.8 µm, single beads can easily be detected in the forward/sideward scatter gate of the flow cytometer.

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Workflow of the multiplex bead-based assay of the MACSPlex Exosome Kit, human. Isolated exosomes are incubated overnight with 39 differently labeled beads, each coupled to a different capture antibody. Bound exosome are detected with APC-conjugated antibodies.



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Flow cytometry analysis of exosome using the MACSPlex Exosome Kit. Example of analyzed exosomes showing (A) gating according to bead size, (B) discrimination of differently labeled bead populations, and (C) measurement of signal intensities of the single bead populations.

The 39 bead populations are distinguished by different fluorescence intensities detected in the B1 and B2 channel of the MACSQuant® Analyzer and MACSQuant® Analyzer 10. The recorded APC signal intensity of each population represents the number of vesicles bound to the respective bead type. The gained surface protein profile of the vesicle population can be used to characterize and compare exosome populations from, e.g., different cell types, stimulated vs. non-stimulated or treated vs. non-treated cells.

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Median signal intensity of the markers represented in the MACSPlex Exosome Kit. Signal intensity was analyzed with the Express Mode of a MACSQuant® Analyzer.

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