Application protocol

Isolation of tumor cells from mouse tumor tissue samples

In this application protocol, we describe how to isolate untouched mouse tumor cells in one easy workflow that improves sensitivity and reduces bias in downstream applications, including next-generation sequencing and tumor cell culture. Mouse solid tumor tissue is dissociated into a viable single-cell suspension and untouched tumor cells are then isolated using MACS® Technology.

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Protocol

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1. Dissociation of tumor tissue

Create a viable single-cell suspension from a solid mouse tumor using the gentleMACS™ Octo Dissociator with Heaters in combination with the Tumor Dissociation Kit, mouse. Follow the protocol from the kit data sheet.

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Tumor Dissociation Kit, mouse

2. Isolation of tumor cells

Isolate untouched mouse tumor cells using the Tumor Cell Isolation Kit, mouse, and LS Columns. Follow the protocol in the kit data sheet.

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Tumor Cell Isolation Kit, mouse

We recommend filtering the magnetically labeled cell suspension to guarantee it is single-celled before separating it on the column.

Place a Pre-Separation Filter (70 µm) on the LS Column.
Rinse the column 3 times with PBS, ensuring that the filter is pre-wetted.
Apply the cell suspension and PB buffer to the filter on the column.

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General workflow for the rapid isolation of untouched tumor cells. The procedure is based on the comprehensive depletion of cells of non-tumor origin by combining automated tissue dissociation and magnetic cell isolation. A negative selection strategy enables the isolation of the tumor cell population without specific knowledge of surface marker expression on these cells. Even from samples containing low numbers of tumor cells, the procedure achieves high isolation purity (>95%) in less than 20 minutes.

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Flow cytometry analysis of isolated tumor cells from three different syngeneic mouse tumors. The antibody cocktail of the Tumor Cell Isolation Kit, mouse, depletes unwanted non-tumor cells, so cell isolation is independent of tumor cell–specific surface markers. Therefore, tumor cells can be isolated regardless of the tumor entity, as shown for the isolation of tumor cells from different mouse tumors induced by GFP-expressing cell lines.

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Cultures of isolated tumor cells remain nearly pure after seven days. Upon magnetic separation, the original bulk (left) and isolated tumor cell (right) fractions were cultured for 3 to 7 days, fixed, and stained. Syngeneic mouse tumor cells were detected by tumor cell–specific GFP expression and fibroblasts were stained with alpha-smooth muscle actin.

References and resources

References

Additional resources

The following is a list of additional resources that may be of interest:

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Materials

Reagent and instrument requirements

For tumor tissue dissociation

Tumor Dissociation Kit, mouse (# 130-096-730)
gentleMACS™ Octo Dissociator with Heaters (# 130-096-427)
gentleMACS C Tubes (# 130-093-237)
RPMI 1640 (# 130-091-440) or DMEM (# 130-091-437) culture media
MACS SmartStrainers (70 µm) (# 130-098-462)
MACSmix™ Tube Rotator (# 130‑090‑753) in combination with an incubator at 37 °C
(Optional) Red Blood Cell Lysis Solution (10×) (# 130-094-183)
(Optional) MACS Tissue Storage Solution (# 130-100-008)
(Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes

For isolation of tumor cells

Tumor Cell Isolation Kit, mouse (# 130-110-187)
QuadroMACS™ Starting Kit (LS) (# 130-091-051)
Pre-Separation Filters (70 µm) (# 130-095-823)
PB Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA) by diluting MACS BSA Stock Solution (# 130‑091-376) 1:20 with PBS. Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column. Always use freshly prepared buffer. Do not use autoMACS® Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.
(Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells
(Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., CD44-VioBlue® (# 130-116-531) and Anti-Ter119-PE (# 130-112-909). Learn more about our antibodies and dyes.
(Optional) Labeling Check Reagent conjugated to, e.g., APC (# 130-122-219) to evaluate purity of sorted cells
PBS buffer

Good to know

Automated protocol

The materials and methods described in this application protocol are for manual processing. Automated cell isolation can be performed with the autoMACS® Pro or the multiMACS™ Cell24 instruments.

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130-098-462