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.

The details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Preparation of sample cells
<ol>
	<li>Culture DF-1 (immortal chicken embryo fibroblasts) cells in six-well plates (5 x 105 cells per well) with Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 38 &#176;C.</li>
	<li>When the cells reach 80% confluence, discard the seru...

Quantifying Pyroptosis in Infectious Bursal Disease Virus-Infected DF-1 Cells

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F., Rodr&#237;guez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exacerbated+apoptosis+of+cells+infected+with+infectious+bursal+disease+virus+upon+exposure+to+interferon+alpha.">Exacerbated apoptosis of cells infected with infectious bursal disease virus upon exposure to interferon alpha.</a> J Virol. 92 (11), (2018).</li><li>Duan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+upregulation+of+chicken+microRNA-16-5p+expression+in+DF-1+cells+following+infection+with+infectious+bursal+disease+virus+(IBDV)+enhances+IBDV-induced+apoptosis+and+viral+replication.">Epigenetic upregulation of chicken microRNA-16-5p expression in DF-1 cells following infection with infectious bursal disease virus (IBDV) enhances IBDV-induced apoptosis and viral replication.</a> J Virol. 94 (2), e01724-e01819 (2020).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+for+voltage-dependent+anion+channel+2+in+infectious+bursal+disease+virus-induced+apoptosis+in+host+cells+via+interaction+with+VP5.">Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.</a> J Virol. 86 (3), 1328-1338 (2012).</li><li>Rodr&#237;guez-Lecompte, J. C., Ni&#241;o-Fong, R., Lopez, A., Frederick Markham, R. J., Kibenge, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+bursal+disease+virus+(IBDV)+induces+apoptosis+in+chicken+B+cells.">Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells.</a> Comp Immunol Microbiol Infect Dis. 28 (4), 321-337 (2005).</li><li>Wang, S., Liu, Y., Zhang, L., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+monitoring+cancer+cell+pyroptosis.">Methods for monitoring cancer cell pyroptosis.</a> Cancer Biol Med. 19 (4), 398-414 (2021).</li><li>Zhang, Y., Chen, X., Gueydan, C., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+changes+during+programmed+cell+deaths.">Plasma membrane changes during programmed cell deaths.</a> Cell Research. 28 (1), 9-21 (2018).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyroptosis+is+driven+by+non-selective+gasdermin-d+pore+and+its+morphology+is+different+from+mlkl+channel-mediated+necroptosis.">Pyroptosis is driven by non-selective gasdermin-d pore and its morphology is different from mlkl channel-mediated necroptosis.</a> Cell Research. 26 (9), 1007-1020 (2016).</li></ol>

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL round-bottom polystyrene tube (12 &#215; 75 mm)</td>
			<td>Corning Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 antibody labeling kits</td>
			<td>Thermo Fisher Scientific</td>
			<td>A20186</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-chGSDME-CT McAb</td>
			<td>SAE Biomedical Tech Company (Zhongshan, China)</td>
			<td>EU0228</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-chGSDME-NT McAb</td>
			<td>SAE Biomedical Tech Company (Zhongshan, China)</td>
			<td>EU0227</td>
			<td></td>
		</tr>
		<tr>
			<td>CellQuest software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic Freezing Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)&#160;</td>
			<td>Gibco by Life Technologies</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F0193-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>FACSCalibur</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Staining Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>00-4222-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Qiu-jing Biochemical Reagent &#38; Instrument Company (Shanghai, China)</td>
			<td>YX-JSB52</td>
			<td></td>
		</tr>
		<tr>
			<td>IBDV Lx strain</td>
			<td></td>
			<td></td>
			<td>IBDV Lx strain was kindly provided by Dr. Jue Liu, Beijing Academy of Agriculture and Forestry, Beijing, China</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Chongqing Photoelectric Instrument Company</td>
			<td>XDS-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse IgG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>M&#38;C&#160; Gene Technology</td>
			<td>CC017</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide(PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.25%</td>
			<td>M&#38;C&#160; Gene Technology</td>
			<td>CC008</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Oscillator</td>
			<td>MIULAB</td>
			<td>MIX-28+</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of pyroptosis by flow cytometry

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 &#60; 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.

Author Spotlight: Flow Cytometric Determination of Pyroptosis in Avian Cells

Examination of Pyroptosis by Flow Cytometry

The objective of our research team is to determine whether avian cells undergo pyroptosis similar to that of mammalian cells. Furthermore, we aim to determine pyroptosis through more accurate and easier means. Many genes in avian species significantly differ from those in mammals, making it difficult to use reagents commonly employed in mammalian cell studies for avian cell death research.Thus, the research on avian cell death is much more difficult than that on human or murine cells. We utilize the characteristic plasma membrane pores in pyroptotic cells formed by gasdermin N-terminal fragments to develop a flow cytometric-based strategy for the determination of pyroptosis. By this novel approach, we observed the occurrence of infectious-bursal-disease-induced pyroptosis in avian cells.In contrast to previous methods such as western blot or AFM analysis, our protocol is able to accurately quantify the percentage of pyroptotic cells by two parameters concurrently, the plasma membrane pore formation and the plasma membrane rupture, facilitating to differentiate cells with pyroptosis from other forms of cell death.

chGSDME-NT on the membrane of DF-1 cells with IBDV infection could be readily detected by flow cytometry 
One of the most important features of pyroptotic cells is the formation of membrane pores by GSDM-NT fragments generated from Gasdermin cleavage. Therefore, pyroptotic cells could theoretically be detected by flow cytometry via examining GSDM-NT on cell membranes using specific antibodies. Thus, DF-1 cells were infected with IBDV, and the pyroptotic cell...

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).

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated ...

examination-of-pyroptosis-by-flow-cytometry

Research

JoVE Journal

Immunology and Infection

Flow Cytometric Analysis for Pyroptosis Studies in Infectious Bursal Disease Virus-Infected Cells

To begin culture five times 10 to the fifth DF-1 cells per well in a six well plate with DMEM supplemented with 10%FBS. When the cells reach 80%confluence, discard the serum containing cell culture medium. After washing the cells three times with PBS, add two milliliters of serum-free cell culture medium, followed by IBDV virus solution to each well.After one hour of virus absorption, wash the cells with PBS three times and incubate them for 24 hours with two milliliters of DMEM supplemented with 2%FBS. Add 500 microliters of trypsin per well, followed by two milliliters of DMEM with 10%FBS after one minute. Centrifuge the cells and remove the supernatant carefully.After resuspending the cell pellet in PBS, gently vortex the tube for three seconds and centrifuge the cells as described earlier. Using a hemocytometer under a microscope, count the cells to resuspend them in an appropriate volume of flow cytometry staining buffer and vortex the suspension. Add 100 microliters of the single cell suspension into a five milliliter round-bottom polystyrene tube.Incubate the IBDV infected cells and mock control cells with one microgram of appropriate antibody on ice for 30 minutes in the dark. Gently vortex tube every 10 minutes. Wash the cells with one milliliter of flow cytometry staining buffer and centrifuge the tube.After discarding the supernatant, resuspend the pellet in 100 microliters of flow cytometry staining buffer. Then incubate the cells with 10 microgram per milliliter of propidium iodide for 10 minutes at room temperature in the dark. And add 400 microliters of flow cytometry staining buffer for the flow cytometry assay.Perform flow cytometry with the blank control tube followed by the single stained tubes to adjust the voltage and compensation parameters before running all the samples. Flow cytometry indicated that a considerable number of cells were positive for N-terminal fragments of chicken Gasdermin E after IBV infection, whereas the C-terminal fragments of cleaved chicken Gasdermin E were barely detectable by flow cytometry using membrane surface antigen staining. The population of N-terminal fragments of chicken Gasdermin E and propidium iodide double-positive cells in IBDV infected cells was significantly greater than that of mock infected controls, suggesting that this part of IBDV infected cells was undergoing pyroptosis.

494 Views.  National Key Laboratory of Veterinary Public Health Security.  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).

Quantifying Pyroptosis in Infectious Bursal Disease Virus-Infected DF-1 Cells

Summary