Dec 16, 2024
Isolating Stem Cells from Mouse Hair Follicles: A Tool for Regenerative Research
- 1Avantyx
- Thomas S Lisse: Correspondence: tlisse@avantyxpharma.com
Protocol Citation: Thomas S Lisse 2024. Isolating Stem Cells from Mouse Hair Follicles: A Tool for Regenerative Research. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3qqwqv25/v1
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: December 15, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 115568
Keywords: Hair follicle, stem cells, Hair follicle stem cells, bulge, regeneration, hair cycle, FACS
Abstract
The hair follicle (HF) is a highly dynamic
organ that serves as an ideal model for studying adult stem cells (SCs). The
permanent portion of the HF, known as the bulge, houses multipotent stem cells
that play a crucial role in tissue homeostasis and regeneration. These
bulge-derived stem cells can differentiate into various cell types, including
interfollicular epidermis, sebaceous glands, and corneal epithelial cells,
under appropriate conditions. Here, we present a detailed protocol for the
isolation and enrichment of hair follicle stem cells (HFSCs) from mouse hair
follicles using fluorescence-activated cell sorting (FACS). This method employs
specific markers such as CD34 and integrin alpha 6 (α6) to reliably identify
and isolate HFSC populations. The protocol begins with the dissection and
enzymatic digestion of mouse skin to release individual hair follicles,
followed by preparation of a single-cell suspension. FACS is then utilized to
separate HFSCs based on marker expression, yielding a highly enriched cell
population suitable for downstream applications. These cells can be used for in
vitro studies to investigate mechanisms of stem cell multipotency,
quiescence, and activation, as well as in vivo grafting models to study
their regenerative potential.
Attachments
Guidelines
To conduct this protocol, strict adherence
to the recommendations and standards outlined and approved by the Institutional
Animal Care and Use Committee (IACUC) guidelines is essential. These guidelines
are designed to ensure the humane treatment, welfare, and ethical use of
animals in research.
Materials
Animals
Item Vendor Cat. Nr.
C57Bl/6J mouse strain The Jackson Laboratory Stock no. 000664
Reagents and labware
Item Vendor Cat. Nr.
5K52 mouse diet (6% fat) LabDiet 5K52
0.25% trypsin-EDTA Thermo Fisher Scientific 25200072
Chelex‱-100 BioRad 1422822
Fetal bovine serum Thermo Fisher Scientific A5669701
Dulbecco's
phosphate-buffered saline Thermo Fisher Scientific 14190144
calcium and magnesium
free
William's E medium, no phenol red Thermo Fisher Scientific A1217601
Penicillin-streptomycin (10,000 U/ml) Thermo Fisher Scientific 15140148
Amphotericin B Thermo Fisher Scientific 15290026
40 µm cell strainer VWR 76327-098
70 µm cell strainer VWR 76327-100
Nunc‱ cell-culture treated multidishes Thermo Fisher Scientific 140675
(6 well)
Nunc‱ cell-culture treated multidishes Thermo Fisher Scientific 142475
(24 well)
35-mm cell culture dish Thermo Fisher Scientific 153066
Nalgene‱ rapid-flow‱ sterile Thermo Fisher Scientific 09-741-03
0.2µm PES disposable filter units Thermo Fisher Scientific 566-0020
CellPro‱ disposable sterile Alkali Scientific VR500/2000
solution bottles
Falcon‱ round-bottom polystyrene FACS StemCell Tech. 38025
tubes, 5 mL
Falcon‱ 50 ml centrifuge tubes Thermo Fisher Scientific 352070
Universal mycoplasma detection kit ATCC 30-1012K
Disposal scapples Thermo Fisher Scientific S17800
Sylgard dissecting plate LS Instrumentations DD-90
Disposable dissecting board Mopec BC007
Microdissection scissors Fine Science Tools 15000-00
Forceps Fine Science Tools 11252-23
Scissors Fine Science Tools 14060-09
Isofluorane solution Covetrus 029404
Collagenase A Sigma-Aldrich 10103578001
Dispase II Sigma-Aldrich 4942078001
Mouse Seroblock FcR BioRad BUF041
DAPI, dihydrochloride Sigma-Aldrich 268298-10MG
Instruments and Software
Item Vendor Cat. Nr.
Technilab BMI BV water acidification system Tecniplast
QuantStudio 3 real time instrument Thermo Fisher Scientific
BD FACSAria II cytometer BD Biosciences
FACSDiva (ver. 8.0.2) BD Biosciences
PrimoVert inverted microscope Zeiss
Countess 3 automated cell counter Thermo Fisher Scientific
ARCO‱ SE small lab animal clipper Kent Scientific
CL8786-KIT
Benchmark B1201 micro bead sterilizer Marshall Scientific B1201
TEC 3 isoflurane vaporizer Covetrus 067512
BSL2 laminar flow hood Thermo Fisher Scientific
1300 Series A2
CO2 incubator Thermo Fisher Scientific
NAPCO Series 8000
Centrifuge Rotina 35 Hettich
Antibodies
Item Vendor Cat. Nr.
CD34-FITC Life Technologies 11-0341-82
CD49f (Integrin α6)-PE/Cyanine7 BioLegend 313621