Research Methods for Cell Pyroptosis

Detailed Guideline on Pyroptosis Research

Pyroptosis research involves several key experimental techniques to study this form of programmed cell death. Scanning electron microscopy (SEM) provides detailed observations of the characteristic membrane rupture and morphological changes that occur during pyroptosis. Western blot (WB) can be used to detect pyroptosis-related proteins such as gasdermin D and inflammasome components. Enzyme-linked immunosorbent assay (ELISA) helps quantify inflammatory cytokines like IL-1β and IL-18, which are crucial markers of pyroptosis. Immunofluorescence (IF) microscopy is useful for visualizing cellular localization of pyroptotic markers. These methods together offer a comprehensive understanding of pyroptosis mechanisms and their regulation.

Cell Morphology Detection

Figure 1. Scanning Electron Microscopy Observations of Pyroptotic Cell Morphology ^[1]

Representative SEM images of CHO cells expressing GSDMD-N-HBD*-HA, treated with DMSO or 4-OHT for 1 hour, are shown. Arrows highlight the bubbling characteristic of pyroptotic cells. SEM analysis showed that the dying cells stayed attached to the culture surface and released multiple pyroptotic bodies.

          2. Immunofluorescence Staining

                                                                                                                         Figure 2. Immunofluorescence Staining of GSDMD ^[1]

Deconvolution microscopy was performed on CHO cells expressing various GSDMD-N-HBD*-HA deletion mutants, following a 1-hour treatment with 4-OHT. After treatment, cells were immunostained for HA and counterstained with Hoechst and PI (Scale bar: 5 μm). PI-positive cells were considered dead. Immunostaining results showed that GSDMD-N-HBD*-HA deletion mutants DN1 to DN4 exhibited behavior similar to wild-type GSDMD-N-HBD*-HA.

Cell Detection of Pyroptosis-Related Proteins

The biochemical characteristics of pyroptosis are primarily marked by the formation of inflammasomes, activation of caspases and gasdermins, and the release of numerous pro-inflammatory cytokines.

During pyroptosis, Gasdermin D (GSDMD, 53 kD) is cleaved to produce an active fragment of approximately 30 kD. Similarly, pyroptotic activation involves the cleavage and activation of Caspase-1, Caspase-4, and Caspase-5, leading to the processing of pro-inflammatory cytokines (e.g., IL-1β, IL-18) and the induction of pyroptotic cell death. The activity of these caspases can be confirmed by detecting their enzymatic cleavage products, while the expression levels of pyroptosis-related proteins can be assessed through Western Blot (WB) analysis.

Figure 3. WB Detection of Pyroptosis-Related Targets ^[2]

Caspase-3 activation and GSDME processing were reduced in CASP1/CASP8 double knockout (DKO) cells compared to CASP1 knockout (KO) cells. These findings indicate that caspase-8 plays a key role in initiating GSDME processing during caspase-1-independent pyroptosis.

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       2. ELISA Detection of IL-1β, IL-18, and Other Inflammatory Cytokines

The inflammatory cytokines IL-1β and IL-18 are activated and secreted in a caspase-1-dependent manner during pyroptosis. Interleukin-1β (IL-1β) is a potent endogenous pyrogen that can induce fever, leukocyte migration to tissues, and the expression of various cytokines and chemokines. Interleukin-18 (IL-18) promotes the production of interferon-γ and is essential for the activation of T cells, macrophages, and other immune cells. Both IL-1β and IL-18 play critical roles in the pathogenesis of various inflammatory and autoimmune diseases.

Figure 4. Detection of IL-1β levels in the supernatant using the ELISA method ^[2]

In contrast to IL-1α release, IL-1β release was inhibited in macrophages treated with VX765 or MCC950.

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Recommended Representative Literature on Pyroptosis Research：

1. Chen et al., Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis. Cell Res 26, 1007-1020 (2016).
2. Aizawa et al., GSDME-Dependent Incomplete Pyroptosis Permits Selective IL-1alpha Release under Caspase-1 Inhibition. iScience 23, 101070 (2020).