Methods in cell biology最新文献_第4页

Flow-cytometry-based assay to study CD8⁺ T cell cytotoxicity against tumor cells. 基于流式细胞术的CD8+ T细胞对肿瘤细胞的毒性研究。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-11-25 DOI: 10.1016/bs.mcb.2025.10.016

Alexey V Sarapulov, Marc-Anthony Rodriguez, Roberta Zappasodi

{"title":"Flow-cytometry-based assay to study CD8+ T cell cytotoxicity against tumor cells.","authors":"Alexey V Sarapulov, Marc-Anthony Rodriguez, Roberta Zappasodi","doi":"10.1016/bs.mcb.2025.10.016","DOIUrl":"10.1016/bs.mcb.2025.10.016","url":null,"abstract":"Cancer immunotherapy approaches aim to instruct the immune system to eliminate tumor cells. Immune checkpoint blockade (ICB) therapies, such as anti-CTLA-4 and anti-PD-1, have significantly advanced the field, and other immunotherapeutic modalities are constantly being developed and tested in pre-clinical and clinical studies. One of the most important outcomes of cancer immunotherapy is efficient elimination of tumor cells by cytotoxic CD8+ T cells. Thus, accurate measurement of tumor cell sensitivity to T-cell-mediated killing is crucial for improving treatment development. This paper presents a flow cytometry-based protocol to assess antigen-specific CD8+ T-cell cytotoxicity using ovalbumin (OVA)-specific CD8+ T cells from OT-1 TCR transgenic mice and OVA-presenting tumor cell lines (either transduced with OVA or pulsed with a class-I-MHC-restricted OVA-derived peptide). This method minimizes contribution from non-antigen-mediated killing into readouts by incorporating non-target cells (not expressing/presenting-OVA cells) into co-cultures. The procedure also allows for the evaluation of T-cell-mediated cytotoxicity under various conditions and enables concurrent immunophenotyping of effector T cells and target tumor cells if desired. The protocol can be easily customized, offers advantages over methods that rely on luciferase-expressing tumor cell lines and provides versatility in detecting multiple parameters, making it a useful and valuable tool for advancing cancer immunotherapy research.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"202 ","pages":"253-276"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13390104/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146194852","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Evaluation of lipid peroxidation as a marker of ferroptosis in colon cancer cells. 评价脂质过氧化作为结肠癌细胞铁下垂的标志。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-03-25 DOI: 10.1016/bs.mcb.2026.02.013

María Pilar Corral-Sancho, Berta Buey, Elena Layunta, Eva Latorre

引用次数: 0

Detecting ferroptotic cells using an antibody against hyperoxidized PRDX3. 使用抗高氧化PRDX3抗体检测铁致细胞。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-03-26 DOI: 10.1016/bs.mcb.2026.02.014

Peihao Sun, Weinan Yin, Mengmeng Sheng, Yawen Meng, Kaiwen Han, Zhihao Li, Yanchao Xu, Shaojie Cui, Jin Ye

{"title":"Detecting ferroptotic cells using an antibody against hyperoxidized PRDX3.","authors":"Peihao Sun, Weinan Yin, Mengmeng Sheng, Yawen Meng, Kaiwen Han, Zhihao Li, Yanchao Xu, Shaojie Cui, Jin Ye","doi":"10.1016/bs.mcb.2026.02.014","DOIUrl":"10.1016/bs.mcb.2026.02.014","url":null,"abstract":"Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation. Although its biological and clinical relevance is increasingly clear, routine and specific detection of ferroptosis remains challenging, especially in tissues and clinical specimens. Indirect measurements of ferroptosis such as lipophilic fluorescent probes (C11-BODIPY581/591), reactive aldehydes (MDA/4-HNE), and pharmacological rescue either lack specificity for ferroptosis, or are impractical for in vivo analysis. Recent work has identified hyperoxidized peroxiredoxin 3 (PRDX3), also known as SO2/3-PRDX3, as a ferroptosis-specific biomarker: Under ferroptotic stress, mitochondrial PRDX3 undergoes selective and irreversible hyperoxidation at its catalytic cysteine residue to generate sulfinic/sulfonic derivatives, a reaction facilitating ferroptosis. Building on this biology, here we outline a method that detects ferroptosis in cells and tissues using 5H7c, a rabbit monoclonal antibody recognizing hyperoxidized but not unmodified PRDX3. The approach offers high specificity, broad sample compatibility (cell lines, animal tissues), and platform flexibility (Western blotting, immunohistochemistry, immunofluorescence).","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"206 ","pages":"95-107"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147817092","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Establishment of LPS-induced sepsis and TNF-induced SIRS. lps诱导脓毒症和tnf诱导SIRS的建立。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-01-29 DOI: 10.1016/bs.mcb.2026.01.015

Jianling Liu, Daichao Xu

引用次数: 0

Measurement of plasma membrane rupture via confocal microscopy. 用共聚焦显微镜测量质膜破裂。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-01-29 DOI: 10.1016/bs.mcb.2026.01.010

Laurel B Stine, Fiachra Humphries

{"title":"Measurement of plasma membrane rupture via confocal microscopy.","authors":"Laurel B Stine, Fiachra Humphries","doi":"10.1016/bs.mcb.2026.01.010","DOIUrl":"10.1016/bs.mcb.2026.01.010","url":null,"abstract":"To maintain homeostasis, cells undergo a tightly regulated process called programmed cell death. Some forms of programmed cell death, such as pyroptosis, can elicit a strong inflammatory response by releasing cytokines through small protein pores. The terminal event of pyroptosis in most cells is plasma membrane rupture (PMR), which breaks down large sections of the plasma membrane and emits intracellular contents that can further amplify the inflammatory signal. In opposition to the previous dogma that PMR is a passive event, it was recently discovered that the transmembrane protein, Ninjurin-1 (NINJ1), is the key executor of PMR. Two models of NINJ1-mediated PMR predict that NINJ1 oligomerizes into filaments or ring-like structures to either open large pores or to excise sections of the membrane. Both models underpin how NINJ1 must oligomerize to execute PMR. When at rest, NINJ1 will autoinhibit oligomerization and activation by forming face-to-face dimer-dimer structures on the plasma membrane. Follow-up studies have shown that NINJ1 executes PMR for other forms of programmed cell death, including apoptosis, ferroptosis, and PANoptosis, as well as mechanical cell death. Thus, assessing NINJ1 function directly through quantification of NINJ1 oligomerization is important to expanding our understanding of both programmed and mechanistic cell death. Here, we describe methods to visualize and quantify NINJ1 oligomerization via immunofluorescence imaging of NINJ1 puncta. This protocol enables more precise and accurate measurement of NINJ1 function during PMR, surpassing conventional methods that just quantify PMR by-products.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"205 ","pages":"47-62"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147663012","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Assessing the activity of caspase-activated pannexin-1 channels by flow cytometry. 流式细胞术评估caspase激活pannexin-1通道的活性。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-05-02 DOI: 10.1016/bs.mcb.2026.04.003

Michael B P F Hector, Ivan K H Poon, Jascinta P Santavanond

{"title":"Assessing the activity of caspase-activated pannexin-1 channels by flow cytometry.","authors":"Michael B P F Hector, Ivan K H Poon, Jascinta P Santavanond","doi":"10.1016/bs.mcb.2026.04.003","DOIUrl":"10.1016/bs.mcb.2026.04.003","url":null,"abstract":"Pannexin 1 (PANX1) is a ubiquitously expressed plasma membrane channel that plays an important role in maintaining cellular homeostasis through the release of small signalling metabolites. Under cell death settings, PANX1 channels can become activated by caspase-3/7 cleavage. This leads to the release of chemotactic 'find-me' signals and anti-inflammatory 'good-bye' signals. Additionally, caspase-activated PANX1 channels can regulate the fragmentation of dying cells. Due to the various cellular pathways that PANX1 channels are involved in, this membrane channel has emerged as an attractive target for novel therapeutics. To assess the effectiveness of pharmacological inhibition on caspase-activated PANX1 channels, the cell-impermeable nucleic acid-binding dye, TO-PRO-3 can be used due to its ability to be selectively taken up into apoptotic cells through caspase-activated PANX1 channels. Here we describe a robust flow cytometry-based protocol utilising Annexin A5 and TO-PRO-3 staining to accurately monitor apoptotic cell death and PANX1 channel activity. This approach can be easily adapted to investigate pharmacological compounds that have the potential to inhibit caspase-activated PANX1 channels.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"209 ","pages":"41-53"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148437553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A 3D model for assessing regulated cell death modalities. 用于评估受调节细胞死亡模式的3D模型。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-04-09 DOI: 10.1016/bs.mcb.2025.03.017

Robin Demuynck, Dmitri V Krysko

{"title":"A 3D model for assessing regulated cell death modalities.","authors":"Robin Demuynck, Dmitri V Krysko","doi":"10.1016/bs.mcb.2025.03.017","DOIUrl":"10.1016/bs.mcb.2025.03.017","url":null,"abstract":"Three-dimensional (3D) models more closely represent the in vivo situation and are therefore more relevant models for drug screening. Among the more than twelve regulated cell death (RCD) modalities, immunogenic cell death (ICD) stands out for its ability to initiate efficient anti-tumor immune response. For example, ferroptosis, which can be induced via the inhibition of GPX4 leading to uncontrolled lipid peroxidation, might be immunogenic under certain conditions. It is crucial to identify which cell death modality is induced, as certain cancer types exhibit resistance to specific forms of cell death. However, a major limitation of 3D models is the lack of high-throughput assays, which often require dissociation of the 3D models, potentially leading to misinterpretation of results. Here, we describe a protocol for identifying and quantifying the induction of RCD modalities in 3D models, such as spheroids. This method eliminates the need for tumor spheroid dissociation and is compatible with other screening techniques, including confocal microscopy. This protocol enables high-throughput screening of various cell death inducers in intact 3D models, serving as a crucial first step in the identification of novel inducers and their specificity for particular ICD and RCD types.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"204 ","pages":"81-92"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581747","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

High-throughput screening for cell death inducers and inhibitors. 细胞死亡诱导剂和抑制剂的高通量筛选。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-04-29 DOI: 10.1016/bs.mcb.2026.04.008

Weimin Sun, Daichao Xu

引用次数: 0

Sensitive detection of lysosomal membrane permeabilization using the galectin puncta assay. 凝集素斑点法检测溶酶体膜通透性的敏感性。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-05-12 DOI: 10.1016/bs.mcb.2026.04.002

Konstantinos Danias, Victoria Alexandra Bjerregaard, Marja Jäättelä

引用次数: 0

Utilizing FLAMBE to study the structure-function relationship and regulation of monomeric BAX activation in solution. 利用FLAMBE研究溶液中单体BAX活化的结构-功能关系及调控。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-05-08 DOI: 10.1016/bs.mcb.2026.04.007

Jesse D Gelles, Md Abdullah Al Noman, Jarvier N Mohammed, Jerry Edward Chipuk

{"title":"Utilizing FLAMBE to study the structure-function relationship and regulation of monomeric BAX activation in solution.","authors":"Jesse D Gelles, Md Abdullah Al Noman, Jarvier N Mohammed, Jerry Edward Chipuk","doi":"10.1016/bs.mcb.2026.04.007","DOIUrl":"https://doi.org/10.1016/bs.mcb.2026.04.007","url":null,"abstract":"The BCL‑2 family member BAX is responsible for inducing mitochondrial outer membrane permeabilization (MOMP) and committing a cell to apoptosis. Specifically, native BAX is an inactive cytosolic monomer, which, upon activation by direct activator BCL‑2 family proteins, undergoes intramolecular rearrangements and structural changes, resulting in BAX translocation to the mitochondrial outer membrane, oligomerization, and MOMP - collectively, we refer to this progression of structural conformers as the BAX activation continuum. In vitro BAX activation studies are invaluable tools for investigating BAX structure-function biology and studying cellular and pharmacological modulators of apoptosis; however, such methodologies are commonly limited to endpoint functional phenotypes (e.g., membrane permeabilization) and thus overlook molecular regulation of soluble monomeric BAX. To address this methodological gap, we developed FLAMBE (a fluorescence polarization ligand assay for monitoring BAX early-activation), a solution-based kinetic assay that infers BAX activation via the concomitant dissociation of a labeled BH3 peptide. FLAMBE maintains the benefits of rapid kinetic data generation in an economical microplate format using commercially available reagents without requiring specialized equipment or large quantities of protein. Herein, this protocol describes how to perform FLAMBE assays, the recommended optimization workflow, and a dual-metric parameterization strategy to distill kinetic data for comparative analyses.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"209 ","pages":"1-26"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148437583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0