Cell Death Detectives Part III: Spotlight on Macrophage Pyroptosis – Model Preparation Techniques and Comprehensive Workflow Solutions

Pyroptosis, as an inflammatory form of programmed cell death, has become a research hotspot in fields such as infection, autoimmune diseases, and tumor immunity. Its molecular mechanism is mediated by the Gasdermin (GSDM) protein family, which triggers cell lysis and pro-inflammatory cytokine release through plasma membrane pore formation, exhibiting a "double-edged sword" effect in immune defense and pathological damage.

Macrophages, as key effector cells connecting innate and adaptive immunity, have become a focal point in pyroptosis research. For instance, researchers at the University of Connecticut found that extracellular vesicles (EVs) released from pyroptotic macrophages carry functional GSDMD pore structures. These vesicles can integrate into neighboring cell membranes, inducing secondary cell death and triggering a cascade of amplified inflammation. This mechanism reveals a new pathological basis for domino-like tissue damage observed in acute respiratory distress syndrome (ARDS) and autoimmune diseases. In graft-versus-host disease (GVHD) studies, pyroptosis of donor-derived macrophages has been shown to drive the development of acute GVHD. In oncology, pyroptosis-inducing adaptive agents can reprogram tumor-infiltrating macrophages, significantly enhancing the efficacy of breast cancer immunotherapy.

In recent scientific research projects, basic mechanistic studies, disease-related investigations, and targeted drug development centered on macrophage pyroptosis have become key areas of focus. The establishment of in vitro macrophage pyroptosis models provides a critical tool for exploring the regulatory mechanisms of pyroptosis and its roles in disease.

Experimental Principle

Lipopolysaccharide (LPS) can activate pyroptosis via both classical and non-classical pathways. In the classical pathway, LPS binds Toll-like receptor 4 (TLR4) and activates the NF-κB pathway via MyD88/TRIF adaptor proteins, promoting the assembly of the NLRP3 inflammasome. In the non-classical pathway, LPS directly activates caspase-4/5 (human) or caspase-11 (mouse), leading to GSDMD cleavage and pyroptosis.

Adenosine triphosphate (ATP), a damage-associated molecular pattern (DAMP), acts through purinergic membrane receptors such as P2X7. Once activated, P2X7 forms transmembrane pores, primarily mediating K⁺ efflux, which activates the NLRP3 inflammasome. Activated NLRP3 recruits and activates caspase-1, which then cleaves GSDMD to trigger pyroptosis. (PMID: 37180116 and 33713214)

Experimental Results

Morphological Observation of Pyroptosis in Raw264.7 Cells

Figure 1: Raw264.7 cells were incubated with LPS alone for 16 h. Under the microscope, the cells showed increased granularity but no characteristic pyroptotic ballooning. However, after co-treatment with 5 mM ATP for 2 h, distinct pyroptotic bubbling (indicated by red arrows) was observed.

Typical morphological features of pyroptotic cells: cell swelling and deformation, organelle distortion, formation of pyroptotic bodies, balloon-like protrusions, and plasma membrane pore rupture.

Caspase-1 Activity and ROS Detection in Pyroptotic Raw264.7 Cells

Figure 2: After LPS and ATP co-induction, Raw264.7 cells were incubated with a caspase-1 probe (E-CK-A481) and ROS probe (E-BC-F005), followed by fluorescence microscopy imaging.

Figure 3: LPS and ATP co-treated cells were analyzed using a caspase-1 colorimetric assay kit (E-CK-A381) and flow cytometry with the ROS probe (E-BC-F005). Caspase-1 activity and ROS accumulation were significantly elevated, consistent with the fluorescence microscopy results in Figure 1.

LDH Release and Pro-inflammatory Cytokine Detection in Pyroptotic Raw264.7 Cells

Figure 4: Culture supernatants from LPS+ATP-treated Raw264.7 cells were analyzed using mouse IL-18/IL-6/TNF-α ELISA kits (E-EL-M0730, E-EL-M0044, E-EL-M3063). Results showed significantly increased levels of secreted cytokines IL-18, IL-6, and TNF-α.

Figure 5: LDH levels in the supernatant were measured using the LDH Cytotoxicity Assay Kit (E-BC-K771-M), revealing markedly increased LDH release from pyroptotic cells.

In summary, the synergistic action of LPS and ATP can significantly induce pyroptosis in Raw264.7 cells, activate intracellular Caspase-1 enzyme, promote NLRP3 inflammasome generation, further enhance intracellular ROS accumulation and LDH increase, and promote the release of inflammatory cytokines such as IL-18, IL-6, and TNF-α.

Experimental Protocol

1. Reagent Preparation: Dissolve ATP in sterile PBS to prepare a 5 M stock, filter-sterilize, aliquot, and store at −20 °C. Prepare LPS in sterile PBS and store at −20 °C.

2. Cell Model Setup: Harvest normally growing mouse monocyte-macrophage cells Raw264.7. Adjust cell density to 2.5-5×105/mL using RPMI-1640 complete medium. Seed cells into 24-well or 6-well plates. Place plates in a 37°C, 5% CO₂ incubator for overnight culture (24 h). After overnight culture, observe cell state (cell density should be ~70%). Add LPS to a final concentration of 1-3 μg/mL, mix gently, and continue culturing in the CO₂ incubator for 16 h. Then, add ATP solution to a final concentration of 5 mM to activate the cells for 2 h.

3. Pyroptosis Detection: Collect cell pellets and cell supernatant, and perform relevant indicator detection. (Refer to relevant product manuals for detailed sample prep and testing procedures.)

Note: For additional details on experimental steps or to learn more about pyroptosis/apoptosis detection solutions, please contact our technical support team.

Related Reagents

Additional In Vitro Model References

Common Pyroptosis Inducers and In Vitro Models

Note: The optimal concentration and exposure time of inducers vary between cell types. Conduct gradient testing based on your model system.

References

[1] Burdette B E, Esparza A N, Zhu H, et al. Gasdermin D in pyroptosis[J]. Acta Pharmaceutica Sinica B, 2021, 11(9). DOI: 10.1016/j.apsb.2021.02.006.

[2] Zheng X, Wan J, Tan G. The mechanisms of NLRP3 inflammasome/pyroptosis activation and their role in diabetic retinopathy[J]. Frontiers in Immunology, 2023, 14.DOI:10.3389/fimmu.2023.1151185.

[3][1] Murao A, Aziz M, Wang H, et al. Release mechanisms of major DAMPs[J]. Apoptosis, 2021(Suppl 1). DOI:10.1007/s10495-021-01663-3.