Application protocol

Isolation of xenografted cells from tumors by depletion of mouse cells

During growth phase in vivo, tumor tissue xenografted in mouse model systems is vascularized and infiltrated by cells of murine origin. Such contamination makes molecular downstream analysis, like expression profiling or next-generation sequencing, challenging. In addition, culturing human tumor cells is frequently hampered by murine fibroblasts overgrowing target cells.

In this application protocol, we describe a time-saving workflow that overcomes these limitations and increases sensitivity of downstream analyses. Xenograft tumor tissue is dissociated into a viable single-cell suspension and untouched human tumor cells are isolated using MACS® technology.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

For tumor tissue dissociation

  • Tumor Dissociation Kit, human (# 130-095-929)
  • 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 depletion of mouse cells

  • Mouse Cell Depletion Kit (# 130-104-694)
  • QuadroMACS™ Starting Kit (LS) (# 130-091-051)
  • 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. 
    ▲ Note: 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. 
  • Pre-Separation Filters (70 µm) (# 130-095-823)
  • (Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., CD326 (EpCAM)-PE. Learn more about our antibodies and dyes.
  • (Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells without fixation
  • (Optional) Labeling Check Reagent conjugated to, e.g., APC to evaluate purity of sorted cells
Matching products:

Protocol


View timeline

The following instructions are for manual cell enrichment.