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Human ACE2 Expression in HeLa Cell Line


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Stable HeLa cell line characterized for endogenous expression of human ACE2, the primary cellular entry receptor for SARS-CoV-2. Epithelial, adherent. ACE2 expressed from the native genomic locus — no transgene, no fluorescent tag, no antibiotic selection. 92% ACE2-positive by flow cytometry. Suitable for SARS-CoV-2 pseudovirus entry assays, ACE2 inhibition screening, siRNA knockdown studies, and co-culture with Spike-expressing cells. Note: does not express TMPRSS2. BSL-2.

Storage and Shipping: Cells are shipped as a frozen vial on dry ice. 

Upon Receipt: Frozen Vial: Transfer immediately to liquid nitrogen for long-term storage. 

⚠️ FOR RESEARCH USE ONLY This product is intended for laboratory research purposes only and is not suitable for human or veterinary diagnostic or therapeutic use.
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For Research Use Only
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Internal Reference: KCL-ENG-101

Description
Characterization
Receipt Protocol
Catalog
Storage & Handling
Safety
FAQs
Product Description

⚠ FOR RESEARCH USE ONLY — Not for diagnostic or therapeutic procedures

KCL-ENG-101 is a HeLa cell line (human cervical adenocarcinoma, epithelial, adherent) characterized and validated for endogenous expression of human Angiotensin-Converting Enzyme 2 (ACE2). Unlike the RFP-tagged ACE2 lines in this series, KCL-ENG-101 expresses ACE2 from its native genomic locus without transgenic modification or antibiotic selection — making it the most physiologically representative model for endogenous ACE2 receptor biology.

ACE2 is confirmed at moderate endogenous expression levels: 92% of cells are ACE2-positive by flow cytometry (MFI greater than 2-fold over isotype control), ACE2 mRNA is detected at Ct 26 to 28 by qPCR, and Western blot shows bands at approximately 120 kDa (glycosylated) and 92 kDa (deglycosylated). The line is suitable for SARS-CoV-2 pseudovirus entry assays, ACE2 inhibition screening, ACE2 receptor biology studies, and co-culture models. All lots are quality tested prior to release for ACE2 expression, viability, mycoplasma, and sterility.

Note: HeLa cells do not natively express TMPRSS2. For assays requiring Spike protein priming via TMPRSS2-dependent entry, co-express TMPRSS2 by transient transfection or supplement with exogenous trypsin.

Recommended Medium

DMEM supplemented with 10% heat-inactivated FBS and 1% Penicillin/Streptomycin. No antibiotic selection is required — this line expresses ACE2 endogenously and does not carry a selection marker. Subculture at 80 to 90% confluence using 0.25% Trypsin-EDTA; split 1:3 to 1:6 every 2 to 3 days at 37°C, 5% CO₂.

Applications
SARS-CoV-2 Pseudovirus Entry Assays ACE2 Inhibition Screening Spike / ACE2 Interaction Studies Co-culture with Spike-Expressing Cells ACE2 RNA Interference (siRNA Knockdown) ACE2 Immunoprecipitation Endogenous Receptor Biology Drug Screening (ACE2 Modulators)
Characterization

All lots are quality tested prior to release. ACE2 expression is confirmed at the protein, mRNA, and surface level for each production lot.

ACE2 Expression & Identity
Parameter Result Specification
ACE2 surface expression (flow cytometry) ✓ Positive 92% positive; MFI >2× isotype control (anti-ACE2, clone 171606)
ACE2 mRNA (qRT-PCR) ✓ Detected Ct 26 to 28 (endogenous); GAPDH Ct 18 to 20 as reference
ACE2 protein (Western blot) ✓ Confirmed Band at ~120 kDa (glycosylated) and ~92 kDa (deglycosylated); anti-ACE2 antibody
Post-thaw viability ✓ Pass ≥85% by Trypan Blue exclusion
Passage number at freeze ✓ Confirmed P4 to P6 from original thaw
Additional Surface Markers
Marker Result Notes
Cytokeratin 18 (CK18) ✓ Positive Epithelial lineage marker; confirms HeLa identity
CD13 (ANPEP) ✓ Positive Alternative coronavirus receptor; present in HeLa
HLA Class I ✓ Positive Immune-competent background; useful for T-cell co-culture assays
TMPRSS2 ≈ Low / Negative Not natively expressed in HeLa; co-express or add exogenous trypsin for Spike-primed entry
CD45 ✗ Negative Confirms absence of hematopoietic contamination
Quality Control
Test Result Method
Mycoplasma ✗ Negative PCR and luminescence assay
Sterility — Bacteria & Fungi ✗ Negative Culture; no bacterial or fungal growth

Certificates of Analysis with lot-specific QC data including flow cytometry histograms and qPCR data are available upon request. Contact Kulture Scientific with your lot number.

Receipt Protocol

KCL-ENG-101 is shipped as a cryopreserved vial on dry ice. These are adherent HeLa cells requiring no antibiotic selection. Follow the steps below upon receipt.

❄  Immediate receipt (same day)
1
Inspect the vial upon receipt. Confirm dry ice is present and the vial cap is intact. Do not allow cells to thaw.
2
Transfer immediately to liquid nitrogen vapor phase (−150°C to −196°C) for long-term storage.
⚠ Do not allow cells to thaw during transit or before intended use. Do not store at −80°C or −20°C.
⚗  Thawing & recovery
1
Thaw vial rapidly in a 37°C water bath with gentle agitation. Remove when a small ice crystal remains.
2
Transfer cells dropwise into pre-warmed DMEM + 10% FBS. Centrifuge at 200 × g (approximately 1000 rpm) for 5 minutes to remove DMSO. Aspirate supernatant.
3
Resuspend pellet in complete DMEM + 10% FBS + 1% Pen/Strep. Seed into a T-25 flask and incubate at 37°C, 5% CO₂.
4
No antibiotic selection is required. Culture in complete medium and allow cells to recover before subculturing.
⚠ No selection antibiotic is used with this line. Do not add Puromycin or G418 — this is an endogenous expression line with no selection marker.
Subculture Guidelines

Subculture at 80 to 90% confluence. Remove medium, rinse with PBS, add 0.25% Trypsin-EDTA and incubate 2 to 3 minutes at 37°C. Neutralize with complete medium and split 1:3 to 1:6 every 2 to 3 days. No selection antibiotic is required. Verify ACE2 expression periodically by flow cytometry if extended culture is planned. Full protocols are available on the Kulture Scientific protocol page.

Product Catalog

KCL-ENG-101 is available as a cryopreserved vial containing at least 5×10&sup5; viable cells in cryopreservation medium (90% FBS + 10% DMSO).

Product Cat. No. Format Cell Number
Human ACE2 Expression in HeLa Cell Line KCL-ENG-101 Cryopreserved Vial ≥5×105 cells/vial
Storage & Handling
❄ Store in Liquid Nitrogen ◾ Ships on Dry Ice ⚠ Do Not Store at −20°C or −80°C
  • Storage — store in liquid nitrogen vapor phase (−150°C to −196°C); cells are stable for more than 5 years when cryopreserved correctly
  • Do not store at −80°C or −20°C — neither temperature is suitable for long-term cell storage; ice crystal formation at these temperatures will irreversibly damage cell membranes and compromise viability
  • Temperature sensitivity — minimize time outside cryogenic storage; cells are sensitive to temperature fluctuations
  • Freeze-thaw cycles — repeated freeze-thaw cycles compromise viability and ACE2 expression; thaw only the quantity needed for immediate use
  • Shipping condition — cryopreserved vials are shipped on dry ice; inspect packaging upon receipt and transfer to liquid nitrogen storage immediately
  • Cryopreservation medium — cells are cryopreserved in 90% FBS + 10% DMSO; wear nitrile gloves during thaw procedures and avoid skin contact with DMSO
  • No selection antibiotic required — this is an endogenous expression line; do not add Puromycin or other selection antibiotics during routine culture
  • Certificate of Analysis — lot-specific QC documentation including ACE2 flow cytometry data, qPCR results, viability, and sterility available upon request
Health & Safety Information
⚠ WARNING! This product is of human origin. HeLa cells are known to harbor HPV-18 sequences and are classified as Risk Group 2. Handle all human-derived biological materials using Universal Precautions in accordance with local BSL-2 regulations. Appropriate PPE must be worn at all times, including gloves, lab coat, and eye protection. All waste must be decontaminated with 10% bleach prior to disposal. Safety Data Sheet available at kulturesci.com.
Regulatory Status

For research purposes only. Not for use in diagnostic or therapeutic procedures. This product has not been tested or validated for clinical use. Not for direct administration into humans or animals.

Biosafety Level

This cell line should be handled under Biosafety Level 2 (BSL-2) conditions based on the following characteristics:

  • Human-derived origin — HeLa cells are derived from human cervical adenocarcinoma tissue; all human-derived cell lines require BSL-2 containment under Universal Precautions
  • HPV-18 sequences — HeLa cells are known to harbor integrated HPV-18 sequences; handle accordingly with BSL-2 precautions at all times
  • ACE2 expression — this line expresses the SARS-CoV-2 entry receptor at endogenous levels; if used with viral constructs (e.g., VSV-G pseudovirus) or live SARS-CoV-2, follow institutional BSL-2 or BSL-3 guidelines as appropriate for the viral component

Users must consult their institutional biosafety committee (IBC) prior to use, particularly when this line is used in combination with pseudoviral or live-virus systems.

Biosafety Considerations
  • Human-derived material with HPV-18 — treat as potentially infectious; work exclusively in a certified biosafety cabinet; follow BSL-2 guidelines
  • ACE2 receptor expression — endogenous ACE2 makes these cells susceptible to SARS-CoV-2 Spike-mediated entry in pseudovirus assays; ensure appropriate containment for any viral component used
  • DMSO (cryoprotectant) — present in cryopreservation medium; skin-penetrating solvent; wear nitrile gloves during thawing
  • Waste disposal — decontaminate all cell culture waste with 10% bleach prior to disposal in accordance with local biohazardous waste regulations
Frequently Asked Questions
How is KCL-ENG-101 different from KCL-ENG-301 (Human ACE2 RFP HeLa)?
KCL-ENG-101 expresses ACE2 from its native genomic locus at endogenous levels — no transgene, no tag, no selection antibiotic. KCL-ENG-301 stably overexpresses a C-terminal RFP-tagged ACE2 fusion under a CMV promoter with Puromycin selection. KCL-ENG-101 is the better choice for studies where native ACE2 expression levels, localization, or regulation are important, and where exogenous overexpression or fluorescent tagging could confound results. KCL-ENG-301 is better suited to assays requiring high, uniform expression or direct fluorescence tracking of ACE2.
Does this line require antibiotic selection?
No. KCL-ENG-101 expresses ACE2 endogenously from the native HeLa genome. There is no stable transgene and no selection marker. Do not add Puromycin, G418, or other selection antibiotics to the culture medium. Routine culture in DMEM + 10% FBS + 1% Pen/Strep is all that is needed.
Does this cell line express TMPRSS2?
No. HeLa cells do not natively express TMPRSS2, the serine protease responsible for priming the SARS-CoV-2 Spike protein for membrane fusion. For assays requiring TMPRSS2-dependent Spike-mediated entry, co-express TMPRSS2 by transient transfection or supplement with low-concentration exogenous trypsin. In the absence of TMPRSS2, SARS-CoV-2 Spike-pseudotyped viral entry occurs primarily via the endosomal cathepsin pathway.
How do I confirm ACE2 expression in my lab?
ACE2 surface expression is most conveniently confirmed by flow cytometry using an anti-human ACE2 antibody (e.g., clone 171606 or equivalent). Approximately 92% of cells should be positive with MFI greater than 2-fold over the isotype control. Western blot will show bands at approximately 120 kDa (glycosylated) and 92 kDa (deglycosylated). qRT-PCR with ACE2-specific primers will show Ct 26 to 28 with GAPDH as the reference gene (Ct 18 to 20).
Can I knock down ACE2 in this line?
Yes. Because ACE2 expression is endogenous, siRNA-mediated knockdown is straightforward. Validated siRNA reagents targeting human ACE2 (e.g., Dharmacon L-007002-00 or equivalent) are compatible with standard HeLa transfection protocols. Validate knockdown by qRT-PCR at 48 hours post-transfection. The endogenous expression background in this line makes it well suited to loss-of-function studies that would be difficult to interpret with an overexpressing transgenic line.
Can I store cells at −80°C?
No. Neither −80°C nor −20°C is suitable for long-term cell storage. Ice crystal formation at these temperatures will irreversibly damage cell membranes and compromise viability. Cells must be stored in liquid nitrogen vapor phase (−150°C to −196°C), where they are stable for more than 5 years.
Where can I find the Certificate of Analysis for my lot?
Certificates of Analysis are available upon request. Contact Kulture Scientific at info@kulturesci.com with the lot number printed on your vial label to receive QC documentation including flow cytometry data, qPCR results, viability, and sterility.