Jul 15, 2025
Fetal liver flow cytometry analysis and FACS
- Peng Xu1,2,
- Rubia Isler Mancuso3,
- Diane S. Krause4,
- Pietro De Camilli1,2
- 1Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 3Department of Laboratory Medicine, Yale Stem Cell Center, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 4Departments of Laboratory Medicine and Pathology, Yale Stem Cell Center, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Protocol Citation: Peng Xu, Rubia Isler Mancuso, Diane S. Krause, Pietro De Camilli 2025. Fetal liver flow cytometry analysis and FACS. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7nbrklwz/v1
Manuscript citation:
Defect in hematopoiesis and embryonic lethality at midgestation of Vps13a/Vps13c double knockout mice
Peng Xu, Rubia Isler Mancuso, Marianna Leonzino, Caroline J. Zeiss, Diane S. Krause, Pietro De Camilli
bioRxiv 2025.05.09.653147; doi: https://doi.org/10.1101/2025.05.09.653147
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 12, 2025
Last Modified: July 15, 2025
Protocol Integer ID: 222379
Keywords: fetal liver flow cytometry analysis, specific murine erythroid progenitor, precursors from fetal liver, using flow cytometry, fetal liver, flow cytometry, cell surface markers of cd71, cell surface marker
Funders Acknowledgements:
National Institutes of Health
Grant ID: NS36251
National Institutes of Health
Grant ID: DA018343
Aligning Science Across Parkinson
Grant ID: ASAP-000580
Yale Cooperative Center of Excellence in Hematology
Grant ID: U54DK106857
Abstract
A protocol to analyze and isolate differentiation-stage-specific murine erythroid progenitors and precursors from fetal liver (E12.5) based on cell surface markers of CD71 and Ter119 using flow cytometry.
Materials
CD3e (BD Biosciences Cat# 553060, RRID:AB_394593),
CD4 (BD Biosciences Cat# 553649, RRID:AB_394969),
CD8a (BD Biosciences Cat# 553028, RRID:AB_394566),
CD45R/B220 (BD Biosciences Cat# 553086, RRID:AB_394616),
CD127 (BD Biosciences Cat# 555288, RRID:AB_395706),
CD11b (BD Biosciences Cat# 553309, RRID:AB_394773),
Ly6G/Ly6C (BD Biosciences Cat# 553125, RRID:AB_394641),
CD71-FITC (BioLegend Cat# 113806, RRID:AB_313567),
Ter119-PE (BD Biosciences Cat# 553673, RRID:AB_394986).
Troubleshooting
Set up mouse crossing and collect E12.5 fetal livers
Male and female heterozygous mice were set up for mating in the evening. Males were removed from the cage on the following day, which was considered E0.5 for the embryos of pregnant females. Pregnancy was assessed by monitoring the female's weight.
At E12.5, embryos were removed, and tail clips were used for genotyping.
Fetal livers were isolated using a dissecting microscope to ensure no contamination by surrounding tissues.
Flow cytometric analysis and FACS
For FACS analysis, fetal livers were dissociated into single cell suspensions and passed through a 100µm nylon strainer to eliminate cell clumps and debris.
Percentages of live cells were determined by trypan blue staining.
Cells were then counted and stained with a lineage cocktail (biotinylated antibodies against CD3e, CD4, CD8a, CD45R/B220, CD127, CD11b, Ly6G/Ly6C) followed by streptavidin-PE-Cy7 (Biolegend,Cat#405206),
anti-CD71-FITC, anti-Ter119-PE, and DAPI (BD, Cat#564907).
Samples were analyzed on LSRII and BD FACSAriaII analyzers for flow cytometric
analysis and FACS sorting, respectively
Protocol references
Koulnis M, Pop R, Porpiglia E, Shearstone JR, Hidalgo D,
Socolovsky M. Identification and analysis of mouse erythroid progenitors using
the CD71/TER119 flow-cytometric assay. J Vis Exp. 2011;(54). Epub 20110805.
doi: 10.3791/2809. PubMed PMID: 21847081; PubMed Central PMCID: PMCPMC3211121.