HMC3 NFkB-Luc2

CRL-3304-NFkB-LUC2 ^™

HMC3 NFκB-Luc2 is a luciferase reporter-labeled cell line that endogenously expresses a high levels of PD-L1 and SIRPA. This reporter cell line can be used in cell-based assays for monitoring the activity of NFκB signal transduction pathways.

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Product category: Human cells
Product type: Cell model

Reporter-labeled cell
Organism: Homo sapiens, human
Morphology: Macrophage
Tissue: Brain
Applications: Immuno-oncology

Cancer research
Product format: Frozen
Storage conditions: Vapor phase of liquid nitrogen

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Documentation

Product sheet

Technical data sheet

BSL 2

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Eagle's Minimum Essential Medium (EMEM)

30-2003

Fetal Bovine Serum (FBS)

30-2020

Dimethylsulfoxide (DMSO)

4-X

General

Specific applications: Enabling sensitive and quantitative assessment of signal transduction makes this reporter cell line ideal for high throughput in vitro bioluminescence assays to study signaling pathways and the development of new checkpoint inhibitors for immuno-oncology drug discovery.

Characteristics

Cells per vial: ≥ 1.0 x 10⁶
Growth properties: Adherent
Age: embryo
Antigen expression: PD-L1 and SIRPA verified at ATCC
Comments: HMC3 NFκB-LUC2 luciferase reporter cell line was derived from parental HMC3 (ATCC CRL-3304) cell line by stably expressing firefly luciferase gene (luc2) under control of the nuclear factor kappa B (NFκB) promoter. This cell line was established through lentiviral transduction and single cell cloning. The cells, upon stimulation, express high levels of enzymatically active luciferase protein, which can be detected via in vitro bioluminescence assays. This reporter cell line is useful for monitoring the activity of tumor necrosis factor alpha (TNF-α)-induced signal transduction pathways.

Handling information

Complete medium

The base medium for this cell line is ATCC®-formulated Eagle’s Minimum Essential Medium (ATCC 30-2003). To make the complete growth medium, add the following components to the base medium: fetal bovine serum (FBS; ATCC 30-2020) to a final concentration of 10% and puromycin to a final concentration of 1.0 µg/mL.

Temperature

37°C

Atmosphere

95% Air, 5% CO₂

Handling procedure

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.

Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
Remove the vial from the water bath as soon as the contents are thawed and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio) and dispense into a 25 cm² or a 75 cm² culture flask. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6)
Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

Subculturing procedure

Volumes used in this protocol are for 75 cm ² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #431464) are recommended for subculturing this product.

1. Remove and discard culture medium.

2. Briefly rinse the cell layer with Dulbecco's Phosphate Buffered Saline (D-PBS; ATCC 30-2200) to remove all traces of serum that contains trypsin inhibitor.

3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37 °C to facilitate dispersal. .

4. Add 6.0 to 8.0 mL complete growth medium and aspirate the cells by gentle pipetting.

5. Add appropriate aliquots of the cell suspension to new culture vessels. Cultures can be established at 1-4 X 10⁴ viable cells/cm². Do not exceed 7x10⁴ viable cells/cm².

6. Incubate cultures at 37 °C. .

Interval: Maintain the cell density from 1 X 10⁴ to 2 X 10⁵ cells/cm2. Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended. Medium Renewal: Add fresh medium every 2 to 3 days (depending on cell density).

Reagents for cryopreservation

Complete growth medium without puromycin supplemented with 5% (v/v) DMSO (ATCC 4-X)

Quality control specifications

Bacterial and fungal testing: Not detected
Virus testing: Cytomegalovirus (CMV): Not detected

Epstein-Barr virus (EBV): Not detected

Hepatitis B virus (HBV): Not detected

Human Immunodeficiency virus (HIV): Not detected

Human papillomavirus (HPV): Not detected
Functional tests: Luciferase assay: > 10-fold increase in activity, basal to stimulation

History

Depositors: ATCC
Year of origin: 2023

Legal disclaimers

Intended use

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.

Warranty

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid. Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Material Transfer Agreement Addendum for Screening Applications

For-profit organizations
For every order of this item, you must provide a signed Material Transfer Agreement Addendum for Screening Applications. We cannot ship this item until we receive this addendum. The person signing the addendum as the principal investigator must match the end user as listed on the applicable sales order for the item.

Email the signed addendum to [email protected] with a reference to both your account and sales order numbers. Once received, your addendum will be reviewed, and this item will be released for shipment if all requirements are met. Additional fees may apply if this product is being used for a screening use (ATCC ACS-2103F), and these fees will be applied after your order is confirmed. If you need assistance with your order, please contact our Customer Care team or your applicable distributor.

This material is subject to the following restrictions in addition to those outlined in the ATCC Material Transfer Agreement:

Luciferase Label License

For information on obtaining additional rights, please contact:

ATCC Licensing
Email: [email protected]

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Brasier AR, et al. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7(10): 1116-22, 1989. PubMed: 2698191

Hsu HY, et al. Inhibition of macrophage scavenger receptor activity by tumor necrosis factor-alpha is transcriptionally and post-transcriptionally regulated. J. Biol. Chem. 271: 7767-7773, 1996. PubMed: 8631819

Ollivier V, et al. Elevated cyclic AMP inhibits NF-kappaB-mediated transcription in human monocytic cells and endothelial cells. J. Biol. Chem. 271: 20828-20835, 1996. PubMed: 8702838

Nakagawa Y, Chiba K. Diversity and plasticity of microglial cells in psychiatric and neurological disorders. Pharmacol Thera 154: 21–35, 2015. PubMed: 26129625

Janabi N, et al. Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen. Neurosci Lett 195(2): 105-108, 1995. PubMed: 7478261

View All Curated Citations for this Product

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