Applying flow cytometry‐based isolation of platelet‐derived microparticles to banked human plasma for cellular studies

Ethical approval

All human studies were performed with appropriate ethical approval from Yale University (HIC no. 0603001208) and in accordance with the Declaration of Helsinki [11].

Human plasma preparation

Peripheral blood was collected from patients in 10‐ml EDTA tubes and centrifuged at 1,500 × g for 30 min to separate plasma, which was stored at −80 °C. For fresh platelet‐rich plasma samples (PRPs), whole blood was collected in acid‐citrate dextrose (ACD) tubes and centrifuged at 280 × g for 10 min with no brake, and the PRP layer was collected. Blood bank platelets were obtained after their expiration date from the Yale‐New Haven Hospital Blood Bank (https://medicine.yale.edu/labmed/clinical-service/bloodbank/). For flow cytometry comparison to banked plasma samples, platelets were isolated from fresh PRP by centrifugation and treated with rat collagen, Type 1 (Sigma‐Aldrich, St. Louis, MO, USA, Catalog No.: 08‐115) for 20 h at 37 °C to generate PMPs, as previously described [12]. PMPs were similarly generated from blood bank platelets for experiments.

Staining of PMPs for flow cytometry

A 10‐μl sample of plasma or isolated PMP was diluted with 40 μl phosphate‐buffered saline (PBS), and CD41‐AlexaFluor 647 (BioLegend, San Diego, CA, USA, Catalog No.: 303725) or isotype control was added at a 1:50 dilution. [Correction added on 11 September 2026, after first online publication: In the preceding sentence, ml has been corrected to μl.] Additional antibodies are listed in supplementary material, Table S1. Staining was performed at room temperature in the dark for 30 min. Samples were diluted to a final volume of 1 ml with PBS prior to analysis. For detergent experiments, plasma was treated with 0.2% Triton X‐100 for 30 min prior to antibody staining.

Flow cytometry analysis and sorting

Flow cytometry was performed on a BigFoot™ Spectral Cell Sorter (Thermo Fisher Scientific, Waltham, MA, USA) using a 100‐μm nozzle. Particles were collected in sterile PBS (Gibco, Waltham, MA, USA, Catalog No.: 10010‐023). Size calibration was performed using polystyrene beads (Apogee, Catalogne, Spain, Catalog No.: 1527). Further details on the flow cytometry methods are provided in the Supplementary Materials and Methods.

Sorted PMPs were treated in some cases with 1 μM CellTracker™ Red CMTPX (Invitrogen, Waltham, MA, USA, Catalog No.: C34552) for 15 min at 37 °C. [Correction added on 11 September 2026, after first online publication: In the preceding sentence, mM has been corrected to μM.] Microparticles were then ultracentrifuged at 20,000× g for 90 min at 4 °C and resuspended in 0.5 ml PBS. Alternatively, PMPs were concentrated using Amicon Ultra 15 ml 100 K filters (Amicon, Darmstadt, Germany, Catalog No.: UFC9100) by centrifugation at 3,000 × g for 60 min. PMP suspensions were added to fibronectin‐coated coverslips for microscopy using a Leica SP8 LIGHTNING confocal microscope (Leica, Wetzlar, Germany).

Electron microscopy (EM)

EM grids (200‐mesh copper; EMS, Morgantown, PA, USA) were glow‐discharged at 20 mA for 20 s to render the surface hydrophilic prior to staining. A 5‐μl unfixed aliquot of sample was applied to the grid and incubated for 3 min, then blotted using Whatman filter paper. Without allowing the sample to dry, the grid was transferred onto a drop of 2% uranyl acetate for 1 min, followed by three rapid rinses with HPLC‐grade water (JT Baker, Phllipsburg, NJ, USA). The grid was then thoroughly air‐dried before imaging on an 80‐kV FEI Tecnai G2 Biotwin transmission electron microscope (Thermo Fisher Scientific, Hillsboro, OR, USA) equipped with an AMT NanoSprint15 MK‐II, Config M camera (AMT, Woburn, MA, USA).

Nanoparticle tracking analysis

Nanoparticle tracking analysis (NTA) was conducted using a ZetaView instrument (Particle Metrix, Holly Springs, NC, USA) with a 488‐nm laser. Sorted microparticles were pelleted by ultracentrifugation at 20,000× g for 90 min at 4 °C and resuspended in PBS.

Cell culture with PMPs

Human umbilical vein endothelial cells were obtained from the Yale Vascular Biology and Therapeutics Program (https://medicine.yale.edu/vascular-biology-and-therapeutics/). Cells were cultured in PromoCell Endothelial Cell Growth Medium MV2 (Heidelberg, Germany), (Catalog No.: C‐22022) with 5% fetal calf serum and the medium supplement recommended by the manufacturer [5 ng/ml recombinant human epidermal growth factor, 10 ng/ml recombinant human basic fibroblast growth factor, 20 ng/ml recombinant human insulin‐like growth factor‐1 (Long R3 IGF‐1), 0.5 ng/ml recombinant human vascular endothelial growth factor 165, 1 μg/ml ascorbic acid, 0.2 μg/ml hydrocortisone], and 1% penicillin/streptomycin stock solution. [Correction added on 11 September 2026, after first online publication: In the preceding sentence, mg has been corrected to μg.] Medium was changed every 48 h or when passaged. For experiments, cells were cultured at a density of 1 × 10⁵ cells/ml in a six‐well plate coated with human fibronectin (Sigma‐Aldrich, lot no. 4078547) on cover slips (CellTreat, Ayer, MA, USA, Catalog No.: 229173). THP‐1 cells were obtained from American Type Culture Collection (Catalog No.: TIB‐202, Manassas, VA, USA) and differentiated to a macrophage phenotype using phorbol‐12‐myristate 13‐acetate (PMA) for 48 h. Resuspended microparticles (250 μl) were added to cultured cells and incubated for 1 h at 37 °C. [Correction added on 11 September 2026, after first online publication: In the preceding sentence, ml has been corrected to μl.] Cells were then fixed with 3.7% paraformaldehyde for 15 min in the dark, permeabilized with 0.1% Triton X‐100 for 15 min, and stained with Alexa Flour 647‐conjugated phalloidin (Invitrogen, Catalog No.: A22287). Cells were then mounted with Prolong Gold Antifade with DAPI (Thermo Fisher Scientific, F6057‐20ML) and visualized using a Leica SP8 LIGHTNING confocal microscope (Leica).

Identification of PMPs by flow cytometry

Our workflow is shown in Figure 1A. Previously published PMP isolation methods require fresh platelets, which limits the feasibility of large human studies, as well as in vitro stimuli that may influence results [5, 13]. Our method uses stored plasma and eliminates the need for stimulation. Banked plasma from a healthy donor was thawed and stained for CD41 (platelet marker). Figure 1B uses fresh PRP to set the size range of particles detected. Figure 1C shows specific CD41 staining (absent in the isotype control; supplementary material, Figure S1A) in the expected size range of PMPs. Figure 1D shows the identical particle size of PMPs generated from fresh platelets from the same donor. PMP size was confirmed using stored platelets from the blood bank (supplementary material, Figure S1B). Note that a CD41 antibody not affected by conformational change of the CD41/CD61 complex upon microparticle release should be used, and co‐labeling with Annexin V can be considered for additional discrimination of PMPs.

Flow cytometry validation of PMPs

MIFlowCyt‐EV and MISEV guidelines were utilized for validation [14, 15]. Buffer alone and isotype control showed minimal nonspecific signal (supplementary material, Figure S2A,B). CD41⁺ PMPs were 95% positive for CD9 (MISEV category 1) and negative for ApoB (MISEV category 3) (supplementary material, Figure S2C,D). Treatment with 0.2% Triton X‐100 and disappearance of the CD41⁺ particles confirmed their identity as cell membrane fragments (Figure 2A). A dilution series to control for coincidence [16] confirmed a linear decrease in events with stable median fluorescence intensity with serial dilutions (supplementary material, Figure S3A–D). Staining for leukocyte marker CD45 and red blood cell marker CD235a in the CD41⁺ population (Figure 2B) demonstrated that only 0.32% of microparticles were positive for CD45 (Figure 2C), and 1.44% of microparticles were positive for CD235a (Figure 2D). Sorted PMPs were labeled with CellTracker (Invitrogen), and the resulting fluorescence confirmed that cytosolic glutathione S‐transferase was present in the PMPs, satisfying MISEV category 2 (cytosolic protein) (Figure 3A). These data show that our technique identifies a highly pure (> 98%) population of PMPs for further analysis.

EM and NTA

We performed NTA and EM to confirm the size of the isolated PMPs and to determine whether our population contained a high percentage of larger (> 800–1,000 nm [17]) apoptotic bodies. Confocal microscopy showed particles of the expected size (Figure 3A; supplementary material, S4), which were similar to those generated from collagen treatment of fresh platelet‐rich plasma (supplementary material, Figure S5). NTA demonstrated a size distribution typical of PMPs with very few particles > 800 nm (Figure 3B). Transmission EM in Figure 3C,D demonstrated FACsorted PMPs, approximately 100–400 nm in size, with morphology showing some PMPs side by side forming lines with budding vesicles from individual PMPs. This is most similar to previously reported PMPs from fresh platelets treated with thrombin or collagen [18].

FACsorted PMPs in downstream experiments

It is critical to define the interactions of PMPs with other cells. Previous reports have shown that in vitro‐generated PMPs are internalized by endothelial cells and monocytes. We performed in vitro generation of PMPs and defined their internalization by human umbilical vein endothelial cells (HUVECs) using confocal microscopy (Figure 4A). We similarly labeled FACsorted PMPs, cocultured them with HUVECs, and detected their internalization (Figure 4B). We also differentiated THP‐1 cells from monocytes using PMA and showed similar internalization of in vitro‐generated and FACsorted PMPs (Figure 4C,D).

These results demonstrate an important strength of our method – that PMPs from banked samples can be used for downstream functional analysis of cell–cell interactions. This technique will allow for the study of PMPs from large biobanks and rare diseases.