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.

Protocol

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Materials

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® (# 130-102-443) and Anti-Ter119-PE (# 130-102-336). Learn more about our antibodies and dyes.
  • (Optional) Labeling Check Reagent conjugated to, e.g., APC (# 130-095-237) to evaluate purity of sorted cells
  • PBS buffer
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