Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.
Total time
~4-6 hours per mouse (tissue harvest through cell sorting; clonal expansion requires 1-2 weeks culture)
Model organism
Mouse (murine liver)
Steps
1
Separate parenchymal and non-parenchymal liver cells
Digest whole murine liver tissue to obtain parenchymal hepatocytes and non-parenchymal cell fractions. This enzymatic dissociation step is critical for accessing stem cell populations within the liver microenvironment.
▶ 01:45
2
Perform red blood cell lysis
Apply red cell lysis buffer to remove erythrocytes and improve cell population purity before downstream enrichment. This step reduces background contamination in subsequent flow cytometry.
▶ 04:51
3
Deplete CD45+ hematopoietic cells from non-parenchymal fraction
Use CD45-targeted immunomagnetic depletion to remove hematopoietic lineage cells and enrich for rare stem cell populations. This negative selection step increases CD133+ stem cell representation in the final population.
▶ 05:51
4
Isolate CD133+ cells by flow cytometry sorting
Apply flow cytometric gating to identify and sort CD133-positive liver stem and cancer stem cells into pure populations. Single-cell sorting enables clonal expansion from individual cells.
▶ 07:31
5
Validate sorted cell populations and expand clones
Confirm CD133+ cell identity through post-sort analysis and establish single-cell derived clonal cultures. Representative sorting efficiency and purity data validate the isolation strategy.
▶ 09:03