Examination of Pyroptosis by Flow Cytometry

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 mammals^1,2,3. Due to the genetic deficiency of GSDMD in chickens^4,5, the mechanism of pyroptosis in chickens remains elusive. The Gasdermin family comprises conserved proteins, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB59^3,6. 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 death^7,8. 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 pathways^11,12,13,14. Previous findings have demonstrated that IBDV infection triggers the release of lactate dehydrogenase (LDH), an indicator of lytic cell death^6,15, 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

The details of the reagents and the equipment used in the study are listed in the Table of Materials.

1. Preparation of sample cells

1. Culture DF-1 (immortal chicken embryo fibroblasts) cells in six-well plates (5 x 10⁵ cells per well) with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 5% CO₂ incubator at 38 °C.
2. When the cells reach 80% confluence, discard the serum-containing cell culture medium, wash the cells three times with phosphate-buffered saline (PBS), and then add 2 mL of serum-free cell culture medium to each well.
3. Add the IBDV virus solution (containing calculated MOI) to each well, and set up a mock-infected control by adding the volume of DMEM equal to that of the virus solution.
4. After 1 h of virus adsorption, discard the culture medium, wash the cells with PBS three times, add 2 mL of DMEM supplemented with 2% FBS to each well, and continue cell cultures for 24 h.
5. 24 h post-IBDV infection, perform trypsin treatment (500 µL per well) for 1 min and subsequently neutralize by DMEM supplemented with 10% FBS (2 mL per well). Afterward, the cells must be harvested to prepare single-cell suspensions for flow cytometry.
6. Centrifuge the cells at 400 x g for 5 min at 2-8 °C. Discard the supernatant using a pipette.
7. Resuspend the cell pellet in PBS and gently vortex the tubes for 3 s.
8. Centrifuge the cells as described in step 1.6 (400 x g for 5 min at 2-8 °C).
9. Repeat the wash of cells as described in step 1.7 and step 1.8.
10. Perform cell counting using a hemocytometer under a microscope. Then, resuspend the cells in an appropriate volume of flow cytometry staining buffer (so that the final cell concentration is 1 x 10⁷ cells/mL). Gently vortex the suspensions.

2. Cell staining for flow cytometry

1. Add 100 µL of the single-cell suspensions from step 1.10 into 5 mL round-bottom polystyrene tubes (12 mm × 75 mm). Prepare mock- and IBDV-infected cells for anti-chGSDME-^NT/CT antibody staining, and use normal mouse IgG as an isotype control. Meanwhile, prepare blank control and single stained tubes with IBDV-infected cells.
2. Add 1 µg of either Alexa Fluor 647-labeled anti-chGSDME-^NT McAb, anti-chGSDME-^CT McAb, or normal mouse IgG (as an isotype control) to IBDV-infected and mock-control tubes, respectively. Subsequently, gently vortex the tubes for 3 s.
3. Incubate the stained cells at 2-8 °C or on ice for 30 min in the dark. Gently vortex tubes every 10 min.
4. Wash the cells with 1 mL of flow cytometry staining buffer by centrifugation at 400 x g for 5 min at 2-8 °C. Discard the supernatant using a pipette.
5. Repeat the wash of cells as described in step 2.4.
6. Resuspend the pellet in 100 µL of flow cytometry staining buffer.
7. Stain the cells of the IBDV-infected group and mock-infected group, as well as the PI-single stained tubes, with 10 µg/mL of PI for 10 min at room temperature, followed by adding 400 µL of flow cytometry staining buffer for the flow cytometry assay.

3. Conducting the flow cytometry assay

1. Turn on the flow cytometer to initialize the instrument and launch the software.
2. Begin with running the blank control tube followed by the single stained tubes to adjust the voltage and compensation parameters of the flow cytometer. Utilize FL3 and FL4 fluorescence channels, which emit at 635 nm, for detecting PI and Alexa Fluor 647 fluorophores, respectively.
3. After configuring all the parameters, run all the samples sequentially.

4. Analysis of flow cytometry data

1. Analyze the Alexa Fluor 647 staining of each sample in the FL4 channel histogram.
2. To assess the proportion of double-positive cells in each sample, utilize the four-quadrant dot plot of FL3/FL4.
3. Set positive thresholds based on isotype control IgG/PI dual-stained samples, ensuring that the proportion of double-positive cells remains below 1%. This approach facilitates measuring the proportion of double-positive cells in all samples.

Representative Results

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 cells were examined by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-^NT McAb and PI staining. A considerable number of chGSDME-^NT positive cells were detected by flow cytometry after IBDV infection (Figure 1). In contrast, mock-infected controls or cells stained with isotype control IgG showed minimal Alexa Fluor 647-positive cells. These findings demonstrate the effective and specific detection of membrane-bound chGSDME-^NT by flow cytometry. Of note, as the C-terminal fragments of cleaved chGSDME (chGSDME-^CT) were retained inside the cytoplasm rather than translocated to the plasma membrane like chGSDME-^NT, chGSDME-^CT fragments were barely detectable by flow cytometry using membrane surface antigen staining, which was in agreement with the fact that pyroptosis was associated with membrane-bound chGSDME-^NT fragments.

Pyroptosis in DF-1 cells can be determined by flow cytometry using dual staining of cells with fluorescein-labeled anti-chGSDME-^NT McAb and PI
When cells were dual stained with Alexa Fluor 647-labeled anti-chGSDME-^NT McAb and PI, followed by flow cytometry analysis in the FL3 and FL4 fluorescence channels, pyroptotic cells could be determined by chGSDME-^NT and PI staining being positive. As shown in Figure 2A,B, a considerable number of chGSDME-^NT positive cells with IBDV infection were detected by flow cytometry assays, and the proportion of chGSDME-^NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than that of mock-infected controls (P < 0.001), suggesting that the formation of membrane pores by the chGSDME-^NT was synchronous with the disruption of cell membranes, thereby causing these IBDV-infected cells to undergo pyroptotic cell death. These data clearly show that IBDV infection induces chGSDME-mediated pyroptosis in host cells. Whereas PI single-positive cells may represent other forms of cell death induced by IBDV infection. Therefore, examination of pore-forming chGSDME-^NT by flow cytometry provides a valuable method for distinguishing pyroptotic cells from those undergoing other forms of cell death.

Figure 1: Examination of chGSDME-^NT bound cells by flow cytometry. DF-1 cells were mock infected or infected with IBDV at an MOI of 1. 24 h after infection, cells were harvested and stained with Alexa Fluor 647-labeled anti-chGSDME-^NT /anti-chGSDME-^CT McAb or isotype control IgG, and analyzed with flow cytometry. chGSDME-^NT-staining positive in flow cytometry assay demonstrated the membrane-bound chGSDME-^NT cells with IBDV-induced pyroptosis. Please click here to view a larger version of this figure.

Figure 2: Examination of pyroptosis by flow cytometry using dual staining with chGSDME-^NT and PI. (A) Dot-plot analysis of pyroptotic cells by flow cytometry. DF-1 cells were infected with IBDV at an MOI of 1. 24 h after infection, cells were harvested, stained with Alexa Fluor 647-labeled anti-chGSDME-^NT /anti-chGSDME-^CT McAb and PI, and analyzed with flow cytometry. (B) The percentage of pyroptotic cells in each group in (A) was plotted and statistically analyzed. Two-way ANOVA was employed to analyze statistical significance. The data represented three independent experiments and were presented as means ± the SD. ***P < 0.001. Please click here to view a larger version of this figure.

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 in mammals^1,2,3, has traditionally been assessed through methods like LDH release^6,15, morphological features¹⁶, and detection of GSDM-^NT/GSDM-^CT fragments via Western Blot or IFA assays¹⁷. However, none of these methods accurately quantifies the percentage of pyroptotic cells. This study leverages the characteristic plasma membrane pores in pyroptotic cells formed by GSDM-^NT fragments to develop a flow cytometry-based strategy for their determination.

When cells were dual-stained with chGSDME-^NT-Alexa Fluor 647 and PI and subsequently analyzed via flow cytometry in the FL3 and FL4 fluorescence channels, pyroptosis was discernible via two parameters concurrently: chGSDME-^NT-Alexa Fluor 647 staining indicating plasma membrane pore formation by the N-terminal fragment of cleaved chGSDME, and PI staining indicating plasma membrane rupture. The data demonstrate that the proportion of chGSDME-^NT/PI double-positive cells in IBDV-infected cells (approximately 33%) was significantly higher than in mock-infected controls (P < 0.001).

The experimental method requires several considerations to ensure accuracy and reliability. Firstly, each sample tube used for flow analysis should contain no fewer than 1 x 10⁶ cells to maintain the precision of the flow analysis. Given the multiple washing steps in the protocol, it is advisable to increase the initial number of cells in each tube at step 2.1 accordingly, for example, to 2 x 10⁶ cells. It's crucial to handle cell washing and transferring with extreme caution to avoid unintended disruption of cell integrity. Additionally, keep in mind that cell density, antibody concentration, and the duration of the operation can all influence the optimal parameter settings. Therefore, it's necessary to adjust the parameters and settings of the flow cytometer based on the specific experimental conditions. In this study, the parameters used were as follows: FSC AmpGain was set to 1.40, and SSC voltage was set to 490 V. The gate in the FSC/SSC plot was set to cover all cell populations for subsequent FL3/4-channel analysis. Fluorescence voltages were set to 600 V for the FL3 channel (PI) and 600 V for the FL4 channel (Alexa Fluor 647). Compensation was set to 38.0% between the FL4 and FL3 channels. The flow rate was set to 200 events/s, with an event threshold set at 10,000.

Assisted by the novel approach, this comprehension of the role of GSDME-mediated pyroptosis in the host response to pathogenic infections in chickens and the underlying mechanisms has significantly advanced. Through the use of specific antibodies, we were able to examine pore-forming chGSDME-^NTvia flow cytometry, distinguishing pyroptotic cells from others and facilitating the determination of the proportion of cells undergoing pyroptosis. As a result, this method offers a distinct advantage over Western Blot or IFA assays in detecting pyroptotic cells.

However, the limitation of this protocol lies in using the N-terminal fragment of chicken GSDME in IBDV-infected cells as an experimental model for studying pyroptosis. Nonetheless, this study marks the first successful utilization of flow cytometry for determining pyroptosis.

In summary, the flow cytometry method described here for examining pyroptosis holds great potential for advancing our understanding of the mechanisms behind pyroptosis in both mammals and avians. This method stands as an effective tool that contributes to research on cell death in life sciences.

Materials

Name	Company	Catalog Number	Comments
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+