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Antibody-trapping presents a widespread pitfall for microscopy and genomics in the nucleus

ABSTRACT: Chromatin has a complex 3D structure and diverse binding proteins that coordinate the genome's most essential functions. Many microscopy and genomics technologies that map chromatin proteins and modifications rely on the diffusion of antibodies (Abs) to target epitopes within whole nuclei. Here, we reveal a critical flaw in such methods that arises when Abs become trapped at the edge of nuclear structures and fail to reach internally-positioned epitopes. This “Ab-trapping” results in artifactual peripheral signal that fundamentally distorts the apparent positions of chromatin features across the genome and nucleus. Using computational modelling and experimental validation, we demonstrate that Ab-trapping is caused by a combination of three compounding factors - high epitope abundance, high Ab affinity, and low Ab diffusion rates. Ab-trapping can thus systematically misrepresent the localization of many prevalent chromatin features like histone modifications, transcription factors, nucleolar proteins, and protein tags. We also show this artifact manifests in multiple technologies, including immunofluorescence microscopy, more recent CUT&Tag-seq, and likely any method relying on Ab diffusion. Finally, we outline readily implementable strategies to identify and mitigate Ab-trapping. Combined, our work presents a previously unrecognized yet prevalent artifact in Ab-based chromatin mapping methods and the means to resolve it.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE293376 | GEO | 2026/06/30

REPOSITORIES: GEO

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Project description:Background: Unexplained recurrent spontaneous abortion (URSA) is a distressing pregnancy disorder with no effective medical intervention. As a potential treatment for URSA, human umbilical cord mesenchymal stem cells (hucMSCs) undergo apoptosis and release apoptotic bodies (ABs) shortly after transplantation. However, the medical effects and the exact doses of ABs in the therapy for URSA remain unclear. Methods: Staurosporine (STS) and ultraviolet (UV) induced apoptosis in hucMSCs. Differential centrifugation, transmission electron microscopy, immunofluorescent staining, and flow cytometry were used to isolate and characterize hucMSC-derived ABs (hucMSC-ABs), respectively. Two mouse abortion models (LPS-induced and CBA/J×DBA/2 immune-mediated) mimicked URSA. Flow cytometry, real-time quantitative PCR, immunofluorescence staining, and western blotting were used to analyze macrophage polarization and inflammatory pathways. Untargeted metabolomic analysis was used to detect the metabolic composition of hucMSC-ABs. RNA sequencing analysis was used to detect the transcriptome of macrophages (Macs). Dorsomorphin was used to inhibit the AMPK signaling pathway. Small interfering RNA (siRNA) was used to knock down Gys1 in raw246.7 macrophages. Results: STS- and UV- induced hucMSC-ABs effectively alleviated abortion in both models, depended on Macs. However, excessive dosage of hucMSC-ABs induced pro-inflammatory Macs (pro-inflam Macs), worsening fetal loss. Appropriate doses suppressed the NF-κB pathway in Macs, while high doses activated it. Neither RNase nor trypsin fully abolished hucMSC-ABs' regulatory effects on macrophage polarization. Metabolomics identified linoleamide and palmitic acid (PA) as the dominant hucMSC-AB metabolites (~1:1 ratio). RNA-seq revealed hucMSC-ABs modulated Nfkb1 and Gys1 in Macs, linked to linoleamide. Appropriate doses of the 1:1 mixture of linoleamide and PA induced anti-inflam Macs, while high dosages of the mixture induced pro-inflam Macs. Linoleamide alone activated AMPK, suppressed NF-κB via Gys1, and protected pregnancy, whereas PA induced pro-inflam Macs and promoted abortion. Conclusion: Appropriate doses of hucMSC-ABs induce anti-inflam Macs in the uterus and prevent fetal loss, while high doses promote the polarization of pro-inflam Macs and lead to fetal loss. The effects of hucMSC-ABs in regulating Macs and treating URSA are related to different doses of linoleamide and PA on Macs. Linoleamide activates AMPK and suppresses NF-κB via Gys1. This study revealed the effect and mechanism of hucMSC-ABs intervention in fetal loss and provided a new idea for the treatment of URSA.

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Antibody-trapping presents a widespread pitfall for microscopy and genomics in the nucleus

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