Ferroptosis Cellular Reporter Assay

Notification: Reference #ht80-gb1lc-av can only be ordered together with reference #ht80-gb1lc.

HMGB1-Lucia reporter assay for ferroptotic cell death monitoring

HMGB1::Lucia Ferroptosis release assay

HT1080-HMGB1-Lucia™ cells are designed to monitor ferroptosis, a form of non-apoptotic cell death [1]. This assay is an alternative to the LDH cytotoxicity assay which measures the activity of lactate dehydrogenase (LDH) released upon rupture of cell membrane integrity. 

More details

Upon ferroptotis activation, the cell membrane ruptures and HMGB1::Lucia is released into the extracellular milieu. Levels of HMGB1::Lucia in the supernatant can be readily monitored by measuring the light signal produced after the addition of QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia® luciferase detection reagent (see figure).

Cell death and subsequent HMGB1::Lucia release induced by RSL3, a strong ferroptosis inducer, can be blocked by Ferrostatin-1, a potent ferroptosis inhibitor. Moreover, ferroptosis in this cell line can be assessed using the classic cytotoxic lactate dehydrogenase (LDH) assay (see figures). 

Key features

• Readily assessable HMGB1::Lucia reporter activity
• Convenient readout using QUANTI-Luc™ 4 Lucia/Gaussia
• Highly sensitive to ferroptosis inducers
• Stability guaranteed for 20 passages

Applications

• Therapeutic development
• Inhibitor and activator screening
• Release assay

InvivoGen’s products are for research use only, and not for clinical or veterinary use.

Figures

RSL3 induces HMGB1::Lucia release in a dose-dependent manner. HT1080-HMGB1-Lucia™ cells were incubated with increasing concentrations of RSL3 (0.001 nM - 5 µM). After 48 hours, the induction of cell death was quantified by measuring the levels of HMGB1::Lucia in the supernatant using the QUANTI-Luc™ detection reagent. Data are shown as (A) fold induction over non-induced cells or (B) percentage (%) activity (mean ± SEM).

RSL3 induces LDH release in a dose-dependent manner. HT1080-HMGB1-Lucia™ cells were incubated with increasing concentrations of RSL3 (0.001 nM - 5 µM). After 48 hours, ferroptosis induction was quantified using the lactate dehydrogenase (LDH) assay. Data is shown as a percentage of cell death (mean ± SEM).

Ferrostatin-1 inhibits RSL3-induced HMGB1::Lucia release in a dose-dependent manner. HT1080-HMGB1-Lucia™ cells were incubated with increasing concentrations of Ferrostatin-1 (0.03 nM - 3 µM) for 30 min followed by the addition of the ferroptosis inducer RSL3 (1 µM final concentration). After 48 hours, the inhibition of cell death was quantified by measuring the levels of HMGB1::Lucia in the supernatant using the QUANTI-Luc™ detection reagent. Data are shown as (A) fold induction over non-induced cells or (B) percentage (%) activity (mean ± SEM).

Ferrostatin-1 inhibits RSL3-induced LDH release in a dose-dependent manner. HT1080-HMGB1-Lucia™ cells were incubated with increasing concentrations of Ferrostatin-1 (0.03 nM - 3 µM) for 30 min followed by the addition of the ferroptosis inducer RSL3 (1 µM final concentration). After 48 hours, the inhibition of cell death was quantified using the lactate dehydrogenase (LDH) assay. Data is shown as a percentage of cell death (mean ± SEM).

Specifications

Cell type: Epithelial

Tissue origin: Fibrosarcoma connective tissue

Target: Ferroptosis 

Specificity: Human

Reporter gene: SEAP

Antibiotic resistance: Blasticidin

Growth medium: Complete DMEM (see TDS)

Growth properties: Adherent

Mycoplasma-free: Verified using PlasmoTest™

Quality control: Each lot is functionally tested and validated.

Contents

• 2-5 x 10⁶  cells in a cryovial or shipping flask.
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)

Details

Cell line description

HT1080-HMGB1-Lucia™ cells are derived from the human fibrosarcoma cell line HT-1080, a standard cell model to study ferroptotic cell death [1-2]. HT1080-HMGB1-Lucia™ cells stably express an HMGB1::Lucia luciferase fusion protein in the cytoplasm, in which the C-terminus of the High-Mobility-Group-Protein B1 (HMGB1) is fused to the Lucia® luciferase.

HMGB1 plays a critical role in the stress response not only inside the cell as a DNA chaperone and cell death regulator but also outside the cell as a prototypical alarmin. Indeed, during non-apoptotic programmed or regulated cell death, such as pyroptosis and ferroptosis, HMGB1 is released along with other pro-inflammatory molecules.

Following ferroptosis activation, the cell membrane ruptures and HMGB1::Lucia is released in the extracellular milieu. Levels of HMGB1::Lucia in the supernatant can be readily monitored by using QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia® luciferase detection reagent (see figures).

Ferroptosis overview

Ferroptosis pathway

In 2012, Dixon et al. [1] discovered a novel form of non-apoptotic cell death characterized by iron overload and accumulation of lethal lipid peroxidation [3]. Because iron chelators were shown to block this type of regulated cell death, it was originally defined as iron-dependent and was called "Ferroptosis" [1]. In contrast to apoptosis, necrosis, and autophagy, ferroptosis is characterized by the build-up of lipid reactive oxygen species (ROS) triggering membrane damage. Cells undergoing ferroptosis do not exhibit classic apoptosis-like features, such as chromatin condensation or membrane blebbing. Instead, mitochondrial shrinkage and increased membrane density are often observed [1-3]. 

Ferroptosis is regulated by a complex network involving iron homeostasis, lipid metabolism, and glutathione-dependent oxidative-reductive balance [4-5]. Iron metabolism plays a central role, as excessive intracellular iron promotes the Fenton reaction, generating lethal amounts of ROS that drive lipid peroxidation [5]. Moreover, the enzyme glutathione peroxidase 4 (GPX4) is a key regulator that protects cells from ferroptosis by reducing lipid peroxides. GPX4 activity is dependent on Glutathione (GSH) which is synthesized using cystine imported via the cystine-glutamate antiporter, System Xc−. When System Xc− is inhibited (e.g. by Erastin), cystine uptake decreases, leading to GSH depletion and GPX4 inactivation. As a result, lipid peroxides accumulate in the cells ultimately leading to ferroptotic cell death [3-5]. 

Ferroptosis in disease

Ferroptosis is involved in various diseases, including neurodegenerative disorders, organ injury-related conditions, and cancer [3-4]. In neurodegenerative diseases, such as Parkinson's and Alzheimer's, ferroptosis contributes to neuronal loss through iron deposition in the brain and oxidative stress [3]. It has also been identified as a major pathogenic driver in other organ injury-related disorders including acute kidney injury and COVID-19-induced myocarditis [4]. Studies have shown that using ferroptosis-specific inhibitors can protect against severe tissue damage in these cases [5]. On the other hand, triggering ferroptosis is being explored as a potential therapeutic approach for treating therapy-resistant cancers [1, 3-5]. Certain types of cancer, especially those with high iron levels or deficiencies in GPX4, are particularly sensitive to ferroptosis-inducing substances. Compounds like RSL3 or Erastin hold promise to overcome drug resistance in chemotherapy and immunotherapy [3]. Advancing our understanding of ferroptosis will continue to drive progress in cell death research and therapeutic development.

References

1. Dixon SJ, et al., 2012. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012 May 25;149(5):1060-72.
2. Zheng J, et al. 2021. Sorafenib fails to trigger ferroptosis across a wide range of cancer cell lines. Cell Death Dis. 2021 Jul 13;12(7):698.
3. Du Y, Guo Z. 2022. Recent progress in ferroptosis: inducers and inhibitors. Cell Death Discov. 8(1):501.
4. Sun S, et al., 2023. Targeting ferroptosis opens new avenues for the development of novel therapeutics. Signal Transduct Target Ther. 8(1):372.
5. Li J, et al., 2020. Ferroptosis: past, present and future. Cell Death Dis. 11(2):88.