Flow cytometry analysis of protein expression using antibody-derived tags followed by CITE-Seq.

Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) is a single-cell phenotyping method that uses antibody-derived tags (ADTs) to quantitatively detect cell surface protein expression and generate transcriptomic data at the single-cell level. Despite the increased popularity of this technique to study cellular heterogeneity and dynamics, detailed methods on how to choose ADT markers and ensuring reagent performance in biological relevant systems prior to sequencing is not available. Here we describe a novel and easy-to-use multiplex flow proxy assay in which multiple protein markers can be measured simultaneously using a combination of ADT reagents and dye-oligo conjugates by flow cytometry. Using dye-oligo conjugates with sequences complementary to the ADT reagents, we can achieve specific binding and evaluate protein marker expression in a multiplex way. This quality control assay is useful for guiding ADT marker choice and confirming protein expression prior to sequencing. Importantly, the labeled cells can be directly isolated based on the specific fluorescence from dye-oligo conjugates using a flow cytometry cell sorter and processed for downstream single-cell multiomics. Using this streamlined workflow, we sorted natural killer cells and T cells efficiently using only ADT and dye-oligo reagents, avoiding the possibility of decreased marker resolution from co-staining cells with ADT and fluorescent antibodies. This novel workflow provides a viable option for improving ADT marker choice and cell sorting efficiency, allowing subsequent CITE-Seq.

Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry.

Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms. Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration. Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants.

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.

Nanoparticles for live cell microscopy: A surface-enhanced Raman scattering perspective.

Surface enhanced Raman scattering (SERS) nanoparticles are an attractive alternative to fluorescent probes for biological labeling because of their photostability and multiplexing capabilities. However, nanoparticle size, shape, and surface properties are known to affect nanoparticle-cell interactions. Other issues such as the formation of a protein corona and antibody multivalency interfere with the labeling properties of nanoparticle-antibody conjugates. Hence, it is important to consider these aspects in order to validate such conjugates for live cell imaging applications. Using SERS nanoparticles that target HER2 and CD44 in breast cancer cells, we demonstrate labeling of fixed cells with high specificity that correlates well with fluorescent labels. However, when labeling live cells to monitor surface biomarker expression and dynamics, the nanoparticles are rapidly uptaken by the cells and become compartmentalized into different cellular regions. This behavior is in stark contrast to that of fluorescent antibody conjugates. This study highlights the impact of nanoparticle internalization and trafficking on the ability to use SERS nanoparticle-antibody conjugates to monitor cell dynamics.