Functional diversification of innate and inflammatory immune responses mediated by antibody fragment crystallizable activities against SARS-CoV-2

Severa, Martina; Etna, Marilena Paola; Andreano, Emanuele; Ricci, Daniela; Cairo, Giada; Fiore, Stefano; Canitano, Andrea; Cara, Andrea; Stefanelli, Paola; Rappuoli, Rino; Palamara, Anna Teresa; Coccia, Eliana Marina

doi:10.1016/j.isci.2024.109703

Cited by 1 publication

(1 citation statement)

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“…viruses and EVs) in biological samples. To achieve this, we will use two different types of well-characterized NPs: small EVs (sEVs) isolated from the supernatant of a melanoma cell line, and Lentivirus based Virus Like Particles (VLPs) (14,29). Furthermore, the development of the analysis protocol for the correct identification of the NPs allowed us to transfer it to cell sorter, enabling the purification of specific sEVs.…”

Section: Introductionmentioning

confidence: 99%

Standardised Flow Cytometric Procedure for Deep Characterization of Nanoparticles

Tirelli,

Grasso,

Barreca

et al. 2024

Preprint

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In recent years, there has been growing interest in the identification and quantification of submicron-sized particles (nanoparticles, NPs), such as extracellular vesicles (EVs) and viruses. This is particularly relevant in clinical settings, where they can be used for screening biomarkers or therapeutic agents. Additionally, they are used in virology or environmental monitoring to detect contaminants like micro- and nano-plastics. The analysis of NPs smaller than 200 nm remains a problem linked to the lack of technology capable of giving reliable results. What makes the analysis of NPs difficult is their great heterogeneity in size, composition, biogenesis, and concentration in different biological fluids. Among the different techniques for the accurate study of NPs, Flow cytometry (FCM) stands out as the ideal method. It is characterized by high resolution, low time consumption, non-destructive sampling, and high throughput. In this article, we propose the optimization of FCM procedures to identify, quantify, and purify sEVs and virus like particles (VLPs). The protocol aims to reduce artifacts and errors in sEVs counting, overall caused by the swarming effect. Different threshold strategies were compared to ensure result specificity. Additionally, the critical parameters to consider when using conventional FCM outside of the common experimental context of use have also been identified. Finally, a fluorescent-sEVs sorting protocol was also developed with highly reliable results using a conventional cell sorter.

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Section: Introductionmentioning

confidence: 99%