Исследование пироптоза методом проточной цитометрии
Summary
This article describes the identification of pyroptotic cells using flow cytometry after dual staining with antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI).
Abstract
Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.
Introduction
Pyroptosis is an inflammatory type of programmed cell death that mainly depends on the formation of plasma membrane pores by Gasdermin (GSDM) D in mammals1,2,3. Due to the genetic deficiency of GSDMD in chickens4,5, the mechanism of pyroptosis in chickens remains elusive. The Gasdermin family comprises conserved proteins, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB593,6. Studies have reported that GSDME from teleost fish and ducks is cleaved by caspase-1/3/7 or caspase-3/7 to induce pyroptotic cell death7,8. However, the role of GSDME-mediated pyroptosis in the host response to pathogenic infections in chickens remains to be elucidated.
Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive poultry disease caused by IBDV9. IBDV, a non-enveloped bi-segmented double-stranded (ds) RNA virus, belongs to the genus Avibirnavirus in the Birnaviridae family10. Previous studies by others and our laboratory have shown that IBDV infection induces cell death in host cells via different pathways11,12,13,14. Previous findings have demonstrated that IBDV infection triggers the release of lactate dehydrogenase (LDH), an indicator of lytic cell death6,15, suggesting that IBDV infection induces lytic cell death in host cells. Furthermore, the data show that IBDV-infected cells exhibit morphological features of pyroptotic cell death, including cell swelling with large bubbles blowing from the plasma membrane and propidium iodide (PI)-staining positive, suggesting that IBDV infection induces pyroptosis in cells.
Considering that the formation of membrane pores in pyroptotic cells by the N-terminal fragment of cleaved GSDM (GSDM-NT) is a hallmark of pyroptosis, theoretically, pyroptotic cells could be detected by flow cytometry by examining GSDM-NT on the cell membrane using specific antibodies. In avian pyroptotic cells, the N-terminal fragment of chicken Gasdermin E (chGSDME-NT) forms membrane pores, allowing propidium iodide (PI) to pass through and bind DNA. Thus, the proportion of pyroptotic cells can be detected using flow cytometry, thereby distinguishing pyroptosis from other forms of cell death, such as apoptosis and necrosis. However, the method for examining pyroptotic cells by flow cytometry has not been reported. In this study, DF-1 cells were infected with IBDV, and flow cytometry was conducted to examine pyroptotic cells using monoclonal antibodies (McAb) against the chGSDME-NT fragment (membrane-bound) and PI staining. Surprisingly, pyroptotic cells were effectively detected by flow cytometry. Furthermore, the proportion of pyroptotic cells could be quantified. These findings provide a powerful and effective means to determine pyroptosis.
This article describes a method for examining pyroptosis in IBDV-infected cells by flow cytometry using anti-chGSDME-NT McAb and PI staining. This method can also be extended to other pathogen-infected cells and applied to examine various cell types with pyroptosis, distinguishing them from other forms of cell death.
Protocol
Representative Results
Discussion
This article describes an effective method for examining pyroptosis using flow cytometry, achieved through dual staining of infected cells with Alexa Fluor 647-labeled anti-chGSDME-NT McAb and PI. This approach can also be applied across various cell types to differentiate pyroptosis from other types of cell death, such as apoptosis and necrosis.
Pyroptosis, an inflammatory type of programmed cell death primarily reliant on Gasdermin (GSDM) D-induced plasma membrane pore formation i…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. Jue Liu for his kind assistance. This study was supported by grants from the National Key Research and Development Program of China (No. 2022YFD1800300), the National Natural Science Foundation of China (No. 32130105), and the Earmarked Fund for Modern Agro-Industry Technology Research System (No. CARS-40), China.
Materials
| 5 mL round-bottom polystyrene tube (12 × 75 mm) | Corning Falcon | 352052 | |
| 6 Well Cell Culture Plate | Corning | 3516 | |
| Alexa Fluor 647 antibody labeling kits | Thermo Fisher Scientific | A20186 | |
| Anti-chGSDME-CT McAb | SAE Biomedical Tech Company (Zhongshan, China) | EU0228 | |
| Anti-chGSDME-NT McAb | SAE Biomedical Tech Company (Zhongshan, China) | EU0227 | |
| CellQuest software | BD Biosciences | ||
| CO2 incubator | Thermo Fisher Scientific | 3100 | |
| Cryogenic Freezing Centrifuge | Eppendorf | 5810R | |
| Dulbecco's Modified Eagle Medium (DMEM) | Gibco by Life Technologies | C11995500BT | |
| Fetal Bovine Serum (FBS) | Sigma-Aldrich | F0193-500ML | |
| Flow Cytometer | BD Biosciences | FACSCalibur | |
| Flow Cytometry Staining Buffer | Thermo Fisher Scientific | 00-4222-26 | |
| Hemocytometer | Qiu-jing Biochemical Reagent & Instrument Company (Shanghai, China) | YX-JSB52 | |
| IBDV Lx strain | IBDV Lx strain was kindly provided by Dr. Jue Liu, Beijing Academy of Agriculture and Forestry, Beijing, China | ||
| Inverted Microscope | Chongqing Photoelectric Instrument Company | XDS-1B | |
| Normal Mouse IgG | Santa Cruz Biotechnology | sc-2025 | |
| Phosphate Buffer Saline (PBS) | M&C Gene Technology | CC017 | |
| Propidium Iodide(PI) | Sigma-Aldrich | P4170 | |
| Trypsin-EDTA, 0.25% | M&C Gene Technology | CC008 | |
| Vortex Oscillator | MIULAB | MIX-28+ |
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