Nov 26, 2025
FACS-Based Isolation Progenitor-Like Keratinocyte Subpopulations from HaCaT Cells for Bulk RNA-Seq Profiling
- Tita Lugui Pacheco Colín1
- 1Center for Research and Advanced Studies of the National Polytechnic Institute
Protocol Citation: Tita Lugui Pacheco Colín 2025. FACS-Based Isolation Progenitor-Like Keratinocyte Subpopulations from HaCaT Cells for Bulk RNA-Seq Profiling . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31do7l25/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: November 10, 2025
Last Modified: November 26, 2025
Protocol Integer ID: 232020
Keywords: FACS, immunostaining, RNA extraction, RNA quality, ephitelial cells, RNA-seq, keratinocyte subpopulations from hacat cell, differentiated keratinocyte fractions with high viability, keratinocyte subpopulation, differentiated keratinocyte fraction, like keratinocyte subpopulations from hacat culture, transcriptomic profiling, like keratinocyte subpopulation, rna integrity for downstream bulk rna, downstream bulk rna, suitable for transcriptomic profiling, bulk rna, early keratinocyte commitment, layer dynamics in human epidermal model, human epidermal model, rna integrity, based isolation progenitor, seq profiling, rna quality, hacat cell, isolation progenitor, sorted population
Abstract
This protocol describes a fluorescence-activated cell sorting (FACS) strategy to isolate progenitor-like keratinocyte subpopulations from HaCaT cultures based on cell-surface markers associated with basal, stem-like states (e.g., high CD49f/ITGA6 with distinct CD71 expression). The workflow covers sample preparation, antibody staining, gating, sorting, and post-sort handling under conditions that preserve RNA integrity for downstream bulk RNA-seq.
Using this protocol, users can obtain highly enriched progenitor-like and more differentiated keratinocyte fractions with high viability and RNA quality (RIN ≥ 9). These sorted populations are suitable for transcriptomic profiling of early keratinocyte commitment, proliferation control, and basal-layer dynamics in human epidermal models.
Protocol materials
RNAprotect Cell ReagentQiagenCatalog # 76526
AccuBlue® Broad Range RNA Quantitation KitBiotiumCatalog #31073
High Sensitivity RNA (NR1) BiOptic Inc.Catalog #C105111/C105211
Direct-zol RNA Microprep KitZymo ResearchCatalog #R2062
RNeasy Plus Micro KitQiagenCatalog #74034
Pacific Blue™ anti-human/mouse CD49f AntibodyBioLegendCatalog #313620
APC/Cyanine7 anti-human CD71 AntibodyBioLegendCatalog #334110
Before You Begin
- Brief background: importance of isolating progenitor-like keratinocytes.
Progenitor-like keratinocytes represent the stem- and transit-amplifying compartment of epidermal models and are essential for long-term renewal and controlled differentiation. In the HaCaT cell line, flow cytometry analysis of α6-integrin and CD71 has revealed a discrete α6^bright/CD71^dim subpopulation with clear stem-like features, including high self-renewal capacity, strong clonogenic potential, and elevated expression of core pluripotency regulators such as SOX2, OCT4, and NANOG (Domínguez-Catzín et al., 2017). Isolating this progenitor-enriched fraction is therefore critical to dissect early regulatory events in keratinocyte biology, as it reduces the masking effects of more differentiated cells and enables a more precise characterization of proliferation, commitment, and epidermal homeostasis.
Citation
LINK
- Marker rationale: e.g., CD49f (ITGA6) and CD71 (TFRC) as indicators of basal and transit-amplifying populations
Note
The use of multiple markers is recommended to ensure the characterization of subpopulations such as: CD133, CD19, CD24, CD38
- Key objective: obtain subpopulations with RNA integrity (RIN > 9) suitable for transcriptomic profiling.
Materials and Reagents
1) Cell line and media:
HaCaT cells (a generous gift from Dr. Norbert Fusenig) were grown in culture dishes in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, NY, USA), L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (50 U/ml), and streptomycin (50 μg/ml). Were incubated in a humidified atmosphere with 5% CO2 at 37 °C and maintained in exponential growth phase.
37 °C 5 %
2) Antibodies: Pacific Blue™ anti-human/mouse CD49f AntibodyBioLegendCatalog #313620 APC/Cyanine7 anti-human CD71 AntibodyBioLegendCatalog #334110
Note
The use of online tools such as FluoroFinder is recommended to design the panel that best suits the experiment, costs, and available equipment as the case may be.
3) Buffers:
PBS (for 1L: KCl 0.2g + NaCl 8g + KH2PO4 0.2 g + Na2HPO4 1.42g)
FACS buffer (PBS 1x + 2% FBS)
Marking solution (PBS 1x + 2% FBS)
PBS+ (PBS + 2% FBS + 1% Hepes 1M pH 7.4)
4) Equipment:
Flow cytometer/sorter ( BD FACSAria II)
Refrigerated centrifuge
RNA extraction kitRNeasy Plus Micro KitQiagenCatalog #74034 Direct-zol RNA Microprep KitZymo ResearchCatalog #R2062
RNA Quality High Sensitivity RNA (NR1) BiOptic Inc.Catalog #C105111/C105211 AccuBlue® Broad Range RNA Quantitation KitBiotiumCatalog #31073
Qsep1 Manual.pdf3.3MB
5) Optimal: RNAprotect Cell ReagentQiagenCatalog # 76526
Equipment Setup
- Instrument BD FACSAria II configuration:
nozzle size: 80mm
flow rate: 1.0 - 2.6
flow threshold: max 8,000 events per second
- Laser and filter settings for Pacific Blue/APC Cy7 (or your fluorochromes).
- Compensation setup and controls (unstained, single-stained, FMO).
Sample Preparation
38m
- Cell harvesting and counting
- Staining procedure with incubation time and temperature
Trypsinized cells are centrifuged in 1.5 mL Eppendorf tubes to form a pellet3000 rpm, Room temperature 00:05:00
Blocking: Incubate with 1 mL of FACS solution 4 °C 00:15:00
Centrifuge 00:03:00 3000 rpm, 4°C
Remove supernatant by decanting Room temperature
Resuspend in 100 mL of labeling solution
Note
For the immunostaining and negative controls, the number of cells was always adjusted to one million (as suggested by the fluorochrome protocols).
This protocol is standardized for the following antibody concentrationsCD49F (1:100) CD71 (1:500)
| Tube | Condition | Staining Buffer | Antibody Volume | Total Volume | |
| 1 | Negative control | 100 µL | 0 µL | 100 µL | |
| 2 | CD49f (1:100) | 90 µL | 10 µL | 100 µL | |
| 3 | CD71 (1:500) | 98 µL | 2 µL | 100 µL | |
| 4 | Double (CD49f + CD71) | 88 µL | 10 µL + 2 µL | 100 µL |
Table1. Example of immunostaining conditions for one million cells in each. From here, the volume is scaled up in the double monostaining as needed.
Incubate 4 °C
Gently agitate every 00:15:00 to avoid precipitation.
Centrifuge 00:03:00 3000 rpm, 4°C
Remove supernatant by decanting.
Wash with PBS+ solution. 600 µL
Centrifuge 00:03:00 3000 rpm, 4°C
Resuspend directly in PBS+ 600 µL into flow cytometry tubes
- Viability dye inclusion (if used).
Note
The transfer time between the wet lab and the sorting unit for this protocol is one and a half hours, so the samples (including the collection tubes) were transported on ice with the aid of gel antifreeze. It is recommended that each laboratory verify the stability of its cell line.
38m
FACS Sorting Procesure
- Gating hierarchy (e.g., FSC/SSC → singlets → viable → CD49f/CD71 quadrants).
- Sorting speed, drop delay, and collection settings.
- Target populations (e.g., CD49f^bri/CD71^dim, CD49f^dim/CD71^bri).
- Purity check post-sort.
Post-Sorting Handling and Transport
1) Conditions for transport and time to RNA extraction.
The sorting process can result in a poor prognosis for genomic material extraction, especially RNA; therefore, the RNAprotect Cell ReagentQiagenCatalog # 76526 reagent was used.
The protocol was standardized as follows:
For every 4 mL of volume recovered from the separated cells, 1 mL of RNAprotect Cell ReagentQiagenCatalog # 76526 was added.
This ensured the protection of the RNA.
2) Temperature stabilization and container setup.
Despite using the RNAprotect Cell Reagent, the samples were transported back on ice 4 °C
3) Note that RIN > 9 was consistently achieved under these conditions.
4) Representative RIN trace image
RNA Extraction and Quality Control
- High Sensitivity RNA (NR1) BiOptic Inc.Catalog #C105111/C105211 The conditions of each of the extraction kits were followed.
HaCaT wt and HaCaT No Bri/dim Direct-zol RNA Microprep KitZymo ResearchCatalog #R2062
HaCaT Bri/dim RNeasy Plus Micro KitQiagenCatalog #74034
- Integrity and quantification (RIN, A260/280, A260/230).
AccuBlue® Broad Range RNA Quantitation KitBiotiumCatalog #31073
Expected Results
- Representative gating plots or purity check images.
- RNA integrity examples (with RIN values).
HaCaT WT_HaCaTwt 1_S1S1_R1_RNA.pdf183.6KB HaCaT Bd _Bridim 1_S1S1_R1_RNA.pdf173.9KB HaCaT No Bd _No Bridim 1_S1S1_R1_RNA.pdf179.5KB Troubleshooting
List common issues and fixes:
| Problem | Possible Cause | Solution | |
| Low RNA integrity | Delayed processing | Use RNAlater or maintain at 4°C | |
| Poor separation | Incorrect compensation | Recheck FMO and gating strategy | |
| Cell clumping | Inadequate filtering | Use 40 µm strainers, add EDTA |
Table2
Acknowledgments
MSc. Dámaris Priscila Romero Rodríguez
National Conahcyt Laboratory for Research and Diagnosis by Immunocyte Fluorometry (LANCIDI). National Institute of Respiratory Diseases Ismael Cosío Villegas, Mexico City
Head of the Nucleic Acids Unit. Department of Genetics and Molecular Biology, CINVESTAV, Mexico City.
Citations
Step 1
Domínguez-Catzín V, Reveles-Espinoza AM, Sánchez-Ramos J, Cruz-Cadena R, Lemus-Hernández D, Garrido E. HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes.
https://doi.org/10.1186/s12985-017-0736-2