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 caspa....

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 seru.......

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+