Examination of Pyroptosis by Flow Cytometry

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 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.

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 cell...

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...

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+