Spectral biophysical cytometry with nanosensors reveals remodelling of immune cells in atherosclerosis

Preparation of BSLBs

1,2-Diarachidonoyl-sn-glycero-3-phosphocholine (20:4-20:4 (DAPC)), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (18:1-18:1 Δ9cis (DOPC)), 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine (18:1-18:1 Δ6cis), 1,2-dielaidoyl-sn-glycero-3-phosphocholine (18:1-18:1 Δ9trans), 1,2-dipalmitoleoysl-sn-glycero-3-phosphocholine (16:1-16:1 Δ9cis), 1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine (16:1-16:1 Δ9trans), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 (POPC)) and ldl cholesterol had been from Avanti Polar Lipids. As a primary step, 10 µl of every lipid at 25 mg ml⁻¹ (in chloroform) was dried beneath a mild stream of nitrogen to kind a skinny lipid movie. The dried movie was rehydrated in 1 ml of SLB buffer (150 mM NaCl, 10 mM HEPES, pH 7.4), vortexed briefly and sonicated utilizing a tip sonicator (energy, 3; obligation cycle, 40%) for 10 min to generate small unilamellar vesicles (SUVs). To kind BSLBs, 45 µl of the SUV answer was combined with 5 µl of silica beads (Bangs Labs) in a ultimate quantity of 500 µl SLB buffer. The combination was bath-sonicated at room temperature for 30 min to facilitate bilayer formation on the bead surfaces. The ensuing lipid-coated beads had been washed 3 times with SLB buffer to take away unbound lipids. NR12S and Pro12A had been synthesized as described beforehand^9,10. For stream cytometry evaluation, beads had been incubated with the membrane order probes Pro12A or NR12S (ultimate focus, 250 nM) for 1 min in stream cytometry tubes instantly previous to acquisition on a Cytek Aurora spectral stream cytometer (5 lasers, 64 channels). For imaging, beads had been plated in an 18-well glass-bottom chamber (ibidi) and excited with a 405-nm laser utilizing a Zeiss LSM 780 confocal microscope outfitted with 32-channel spectral detectors.

Drug remedies on CEM cells

CEM cells (ATCC, CRL-2265) had been cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) beneath commonplace circumstances. The next compounds had been used for drug therapy experiments: CK-666 (5 µg ml⁻¹), latrunculin B (1 µg ml⁻¹), methyl-β-cyclodextrin (12 µg ml⁻¹), sphingomyelinase (1/100 dilution of 25 models), oseltamivir phosphate (40 µg ml⁻¹), swainsonine (1 µg ml⁻¹), digitonin (5 µg ml⁻¹) and valinomycin (25 µM) (all from Sigma-Aldrich), and CCCP (20 µM) (Thermo Fisher). For every situation, 3 × 10⁵ cells had been used. If not acknowledged in any other case, all remedies had been carried out in full medium at 37 °C for 30 min–1 h. Following incubation, cells had been washed 3 times with PBS. Cell viability was assessed utilizing BD Horizon Fixable Viability Stain 660 (FVS660, Reside/Lifeless), utilized for 15 min at room temperature, adopted by two PBS washes. For membrane order evaluation, cells had been stained with Pro12A dye (0.6 µl of 100 µM dye in 300 µl PBS for 300,000 cells) for 1 min straight within the stream cytometry tubes instantly earlier than the acquisition. For mitochondrial depolarization measurements, JC-1 dye (Thermo Fisher) was incubated with the cells for 30 min both previous to or throughout drug therapy. For CCCP remedies, JC-1 (0.5 µl of 250 µM dye in 500 µl medium for 300,000 cells) was added over the last 5, 15 or 30 min of drug publicity, whereas for Digitonin, JC-1 was added in the course of the ultimate 5 min of therapy. Cells had been washed twice previous to Reside/Lifeless staining. For membrane potential measurements with CV1 dye (CytoCybernetics), cells had been incubated for 15 min in various ratios of high-potassium buffer (138 mM KCl, 2 mM NaCl, 2 mM CaCl2, 20 mM HEPES, pH 7.5) and low-potassium buffer (2 mM KCl, 138 mM NaCl, 2 mM CaCl2, 20 mM HEPES, pH 7.5), adopted by therapy with valinomycin for five min. The Nernst potential for potassium was calculated by the equation:

the place R is the gasoline fixed, T is the temperature, F is Faraday’s fixed and [K_out] is the focus of potassium within the exterior answer, assuming an intracellular potassium focus of 140 mM. Cells had been then stained with CV1 (0.5 µl of fifty µM dye in 300 µl PBS for 300,000 cells) for 1 min instantly previous to acquisition. For spectral imaging, cells had been plated in an 18-well glass-bottom chamber and imaged utilizing a Zeiss LSM 780, as described beforehand for Pro12A¹¹. Cells with JC-1 had been excited with a 488-nm laser, and 19 detectors had been set, ranging between 494 and 665 nm with 9-nm intervals. Normalized ratios ((monomer − aggregates)/(monomer + aggregates)) had been calculated utilizing wavelengths of 539 nm (monomers) and 592 nm (aggregates). For imaging of CV1, cells had been excited with wavelengths of 633 nm and picked up between 632 and 694 nm.

Drug remedies on PBMCs

PBMCs (coated by moral allow Dnr 2025-01255-01, 2025-02-20) had been remoted from 45 ml of buffy coat obtained from wholesome donors. The buffy coat was diluted 1:1 with PBS and evenly distributed into two 50-ml Falcon tubes. After thorough mixing with a serological pipette, 30 ml of the diluted pattern was rigorously layered over 15 ml of Ficoll-Paque (Sigma-Aldrich) in three separate 50-ml tubes. Samples had been centrifuged at 400g for 25 min with out making use of the brake. The PBMC layer was collected and transferred into contemporary tubes, adopted by two PBS washes (300g, 5 min, brake on). After the ultimate wash, cells had been resuspended in 20 ml PBS, filtered via a 70-µm cell strainer (Thermo Fisher), and centrifuged at 200g for 10 min. The supernatant was discarded, and the cell pellet was resuspended in cryopreservation medium (90% FBS, 10% DMSO). Cells had been aliquoted (1 ml per vial) and saved at –150 °C till use.

For experiments, frozen PBMCs had been thawed by gradual addition of a 10-fold quantity of prewarmed RPMI 1640 medium supplemented with 10% FBS, adopted by centrifugation at 400g for six min. Cells had been processed in Eppendorf tubes at a ultimate focus of 6 × 10⁵ cells per situation. For mitochondrial depolarization assays, cells had been first incubated with JC-1 dye for 30 min at 37 °C, adopted by two PBS washes. Cells had been then handled with CCCP for five min to induce mitochondrial depolarization and washed twice with PBS. For membrane order evaluation, cells had been handled with sphingomyelinase (Sigma-Aldrich) for 1 h at 37 °C and subsequently washed twice with PBS. Whereas antibody staining was carried out after remedies and JC-1 labelling for mitochondrial depolarization, it was executed earlier than Pro12A or/and CV1 labelling.

Following remedies, cells and their untreated controls had been used to organize each single-stained controls and multicolour antibody panels for spectral stream cytometry. Antibody titrations had been optimized based mostly on staining index calculations. Every pattern was both unstained, stained with a single antibody or probe (for spectral unmixing) or with a full antibody cocktail along with probe(s) in staining buffer (Thermo Fisher). Staining was carried out at 4 °C at the hours of darkness for 30 min, adopted by two PBS washes. Cells had been then resuspended in 500 µl PBS, and viability was assessed by including 1 µl of a Reside/Lifeless dye (1:100 dilution), incubating for 15 min at room temperature at the hours of darkness, adopted by two ultimate PBS washes. Cells had been then resuspended in 300 µl PBS in stream tubes for acquisition.

For Pro12A-only experiments, PBMCs had been stained with the next antibodies: anti-human CD3-PerCP-Cy5.5 (clone SK7, BioLegend), CD56-PerCP-eFluor 710 (clone CMSSB, Thermo Fisher), CD4-NovaFluor Yellow 660 (clone SK3, Thermo Fisher), CD197 (CCR7)-PE-Vio770 (clone REA108|TG8, Miltenyi Biotec), CD14-APC (clone M5E2, BioLegend), CD19-Alexa Fluor 647 (clone HIB19, BioLegend), CD11c-APC-R700 (clone 3.9, BD Biosciences), CD45RA-APC-H7 (clone HI100, BD Biosciences), CD8a-APC-Hearth 810 (clone SK1, BioLegend) and Fixable Viability Stain 570 (BD Biosciences). For JC-1-only and Pro12A–JC-1 mixture experiments, the next antibodies had been used: CD3-PerCP-Cy5.5, CD56-PerCP-eFluor 710, CD4-NovaFluor Yellow 660, CD197 (CCR7)-PE-Vio770, CD14-APC, CD11c-APC-R700, CD45RA-APC-H7, CD8a-APC-Hearth 810 and Fixable Viability Stain 660 (BD Biosciences). For CV1-only and Pro12A–JC-1–CV1 mixture experiments, the antibody panel included: CD56-PerCP-eFluor 710, CD19-Alexa Fluor 647, CD45RA-APC-H7 and CD8a-APC-Hearth 810.

PBMCs from people with atherosclerosis

This research was accredited by the Lokman Hekim College Scientific Analysis Ethics Committee (approval quantity 2024/46; approval date 30 January 2024) and by Ethics Assessment Authority Stockholm (Dnr 2025-02560-01, 2025-04-11). Written knowledgeable consent was obtained from donors previous to the research. All procedures involving human contributors adhered to the moral rules outlined within the 1964 Helsinki Declaration and its subsequent amendments or equal moral requirements. PBMC samples had been obtained from sufferers with angiographically confirmed coronary artery illness, however with out prior myocardial infarction, as a part of the inclusion standards. Intercourse- and age-matched wholesome topics had no identified heart problems or drug therapy for it, had by no means smoked, had no historical past of diabetes and had no household historical past of most cancers. Entire blood from 38 sufferers and 26 wholesome controls was drawn into EDTA tubes (Becton Dickinson) and PBMCs had been remoted instantly. No statistical strategies had been used to predetermine pattern sizes. The samples weren’t randomized, and knowledge assortment and evaluation weren’t carried out blind to the circumstances of the experiments to have the ability to measure the identical variety of wholesome and affected person materials on every day. No donors had been excluded. Peripheral blood was diluted 1:1 with 1× DPBS and punctiliously layered over an equal quantity of Ficoll-Paque answer in a 50-ml conical tube, tilted at ∼45°, to keep up part separation. The tubes had been centrifuged at 400g for 30 min at room temperature with minimal acceleration and no brake to protect interface integrity. The mononuclear cell layer (buffy coat) was collected, washed sequentially with 1× DPBS and IMDM medium (Thermo Fisher), and centrifuged at 300g for 10 min between washes. After the ultimate wash, cells had been cryopreserved at 1 × 10⁶ cells per cryovial in freezing medium (90% FBS + 10% DMSO). The cryovials had been cooled at a price of 1 °C min⁻¹ in a Mr Frosty and saved in a single day at −86 °C. All vials had been subsequently transferred to liquid nitrogen till evaluation. The dealing with of the PBMCs and the evaluation pipeline for membrane order and mitochondrial depolarization with Pro12A and JC-1 is described above.

SBC measurements and evaluation

Spectral stream cytometry knowledge obtained with a Cytek Aurora (Supplementary Knowledge 1) had been analysed utilizing FCS Specific 7 (DeNovo Software program), and downstream computational analyses had been performed utilizing Python-based pipelines. Spectral unmixing was carried out inside FCS Specific utilizing unstained, autofluorescence for lymphocytes and monocytes, single-stained controls for every fluorochrome, and probes to precisely resolve sign overlap throughout channels. Spectral signatures had been rigorously curated and reviewed to reduce spillovers and keep optimum separation between fluorochromes.

To establish the optimum fluorescence channel pair for separating experimental circumstances, we computed ratiometric values for all dual-channel combos. Every pair was evaluated utilizing the Kolmogorov–Smirnov statistic and absolutely the distinction in median ratios. An total rating combining each metrics was used to rank channel pairs, with changes for prime intragroup variance. The highest-ranking pair was chosen as probably the most discriminative along with excitation/emission properties of the probes. For BSLBs stained with Pro12A or NR12S, V1–V9 (428–598 nm) and YG2–YG6 (598–697 nm) channels had been used, respectively. For mobile knowledge, Pro12A emission was captured in V1–V7 (428–542 nm), JC-1 in B3–B5 (542–598 nm) and CV1 in B7–R5 (661–738 nm). When Pro12A and JC-1 had been coincubated, V1–V5 (428–508 nm) had been chosen for membrane order quantification. When all probes had been mixed (Pro12A, JC-1, CV1), V1–V5 had been used for Pro12A and B9–R5 (697–738 nm) for CV1. ROUT 1% outlier was utilized to eradicate instrument-related faulty or disproportionately massive values. Gating methods for all circumstances, together with singlet discrimination, avoiding internalized probes, viability gating, and main immune lineage identification, are detailed (Supplementary Figs. 10–15).

To discover high-dimensional spectral options and visualize population-level variations, UMAP was used for dimensionality discount. Uncooked fluorescence depth values had been first normalized on a per-cell foundation by dividing every channel’s worth by the utmost depth throughout all channels for that cell. This per-cell normalization preserved the relative spectral composition whereas mitigating absolute depth variability. The intensities had been then normalized utilizing StandardScaler (scikit-learn), remodeling every characteristic to have zero imply and unit variance. UMAP was carried out utilizing the umap-learn bundle with parameters tuned to steadiness world and native construction preservation (for instance, n_neighbors, min_dist). These parameters had been chosen to keep up separation between biologically significant subpopulations whereas preserving finer construction within the knowledge. For classification and predictive modelling duties, we utilized Excessive Gradient Boosting (XGBoost) through the xgboost Python library. Previous to modelling, enter knowledge had been scaled utilizing StandardScaler and labels had been numerically encoded utilizing LabelEncoder (scikit-learn). Bayesian hyperparameter optimization was performed utilizing the skopt bundle (Bayesian optimization with Gaussian Processes), with search area definitions spanning key XGBoost hyperparameters (for instance, learning_rate, n_estimators, max_depth, subsample, colsample_bytree, reg_alpha, reg_lambda, gamma, min_child_weight). Mannequin efficiency was evaluated utilizing stratified ok-fold cross-validation (usually 10-fold; 5-fold for small pattern units), with classification accuracy and confusion matrices.

scRNA-seq of biophysically chosen PBMCs

PBMC samples (4 atherosclerosis and 4 sex- and age-matched wholesome controls (A. Wholesome)) had been processed for scRNA-seq utilizing the 10x Genomics Chromium Single Cell 3′ v.4 platform (Supplementary Knowledge 2), following the producer’s protocol. Briefly, after thawing and washing, cells had been resuspended in PBS containing 0.04% BSA, counted and assessed for viability. Cell suspensions had been loaded into the Chromium Controller to generate gel bead-in-emulsions, adopted by reverse transcription, cDNA amplification and library development utilizing the Single Cell 3′ v.4 Reagent Package. Remaining libraries had been quality-checked utilizing an Agilent Bioanalyzer and quantified by quantitative polymerase chain response. Sequencing was carried out on an Illumina NovaSeq X Plus system (NovaSeq X Sequence Management Software program v.1.2.2.48004) utilizing a 151 nucleotide (nt) (learn 1) – 10 nt (index 1) – 10 nt (index 2) – 151 nt (learn 2) configuration on a ‘25B’ mode stream cell.

Sequencing knowledge had been processed with Cell Ranger v.9.0.1 utilizing the GRCh38-2024-A reference genome to generate filtered gene-barcode matrices. Downstream evaluation was performed in R v.4.4.3 utilizing the Seurat v.5.2.1 pipeline²⁶. Briefly, commonplace high quality management excluded cells with >20% mitochondrial content material or <5% ribosomal gene expression. Genes resembling MALAT1, mitochondrial (MT-), ribosomal (RPS, RPL), and haemoglobin (HB) genes had been additionally excluded. Pattern-specific anticipated doublet charges (starting from 1.2% to 7.2%) had been utilized utilizing DoubletFinder to establish and exclude potential doublets. The ultimate dataset was normalized, scaled and subjected to PCA and UMAP for dimensionality discount, adopted by graph-based clustering utilizing the Louvain algorithm and GSEA-informed cell-type prediction. To give attention to immune dynamics, we particularly chosen T cells and redefined identities based mostly on illness situation (Atherosclerosis versus A. Wholesome). Differential gene expression evaluation was carried out utilizing the Wilcoxon rank-sum check with conservative filtering (logfc.threshold = 0.25, min.pct = 0.15), figuring out genes considerably up- or downregulated within the illness context. These gene units had been analysed for pathway enrichment utilizing the GO Organic Course of 2023 database through enrichr. Enrichment outcomes had been filtered for pathways containing key phrases related to lipids and mitochondria and visualized with −log₁₀(P worth) bar plots to spotlight condition-specific adjustments in T-cell biology.

Mass spectrometry of biophysically chosen PBMCs

We carried out direct infusion shotgun lipidomics evaluation on chosen atherosclerosis (n = 4) and sex- and age-matched wholesome management (n = 4) samples, utilizing the identical donor materials as for the scRNA-seq evaluation. Previous to lipidomics, main human T cells had been remoted from PBMCs through unfavourable magnetic choice utilizing the MojoSort Human CD3 T Cell Isolation Package (BioLegend), following the producer’s protocol. The ensuing cell populations exhibited >90% purity. Cells had been counted utilizing a BioRad TC20 cell counter and assessed for viability with trypan blue staining. Subsequently, cells had been washed twice with PBS, adopted by a ultimate wash with 140 mM ammonium formate. Cell pellets had been then snap-frozen and saved −80 °C till the evaluation. Previous to sampling, the cells had been quickly thawed and lysed utilizing methanol:H₂O 9:1 v/v (0.1% formic acid), concentrating on a ultimate cell focus of 1000 cells µl⁻¹ in every pattern. The cell lysates had been centrifuged for 1 min at 8,000 r.p.m., and the supernatant was straight analysed by immersing the direct infusion probe²⁷ within the solvent. The electrospray voltage was straight utilized on the metal capillary and was set to 2,150 V. Mass spectrometry was carried out in positive-ion mode on an Orbitrap IQ-X (Thermo Fisher Scientific) instrument the place the AGC goal was 40% and scans had been acquired at 240,000 decision (at m/z 200) within the mass vary of m/z 250–1,200. For every measurement, a 1-min-long direct infusion was carried out, equivalent to 120 particular person mass spectra on common. The acquired RAW knowledge information had been transformed to .mzML utilizing MSConvertGUI (ProteoWizard, v.3.0.22285). Analytes had been recognized based mostly on correct mass match (3 ppm tolerance) to our in-house library utilizing custom-made MATLAB scripts and the total-ion-current-normalized depth of molecular options had been extracted from every spectrum and averaged via all scan occasions. Subsequent, the identities of detected molecules had been confirmed utilizing tandem mass spectrometry measurements on pooled samples. Particularly, we pooled equal volumes of all 4 management and all 4 atherosclerotic samples after which carried out higher-energy collisional dissociation fragmentation utilizing normalized collisional energies of 30, 40 and 50. All tandem mass spectrometry spectra had been manually curated for structural annotation, and within the case of a number of adduct types of the identical molecular characteristic, solely the best depth one was used for additional knowledge evaluation. Sparse partial least squares (PLS) discriminant evaluation was carried out to establish options distinguishing between atherosclerosis and wholesome T-cell lipidomes. Lipid depth values had been standardized first and the highest 30 discriminative options had been chosen utilizing univariate evaluation of variance (ANOVA) (SelectKBest, f_classif). PLS discriminant evaluation was performed utilizing two parts, and the defined variance per element was calculated. Analyses had been performed utilizing Python’s scikit-learn and matplotlib libraries.

Reporting abstract

Additional data on analysis design is offered within the Nature Portfolio Reporting Abstract linked to this text.