Attune nxt acoustic focusing cytometer

To perform flow cytometric detection, CD81-positive exosomes were isolated with Exosome-Human CD81 Flow Detection (from cell culture; Invitrogen, Life Technologies), following manufacturer’s instructions. Briefly, preenriched exosome solution, prepared using Total Exosome Isolation reagent, was added to a tube containing CD81 isolation beads previously washed and incubated at 4 °C overnight with end-over-end mixing. After incubation, the bead-bound exosomes were isolated with a magnetic separator (MagnaRack, Invitrogen) and washed once with Assay Buffer containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich) in DPBS. Then, isolated CD81-positive exosomes were labeled with anti-human CD9-PerCP Cy5.5 or anti-human CD81-PE antibodies (Invitrogen, Life Technologies), and 150 µL of sample were detected with flow cytometer (Attune™ NxT Acoustic Focusing Cytometer, Life Technologies). Negative control was performed by repeating entirely the previously described staining procedure with DPBS (vehicle) instead of exosomes. Data collected from the experiments were analyzed using Attune NxT Software version 2.5 (Life Technologies). At least 3 independent experiments were performed for each point.

HEK293T cells were cultured in 24-well plates (2.5 × 10⁵ cells per well) for 24 h, and incubated for 12 h with 10 μM CPP-D7, CPP-D3, D7, D3, or an equivalent volume of PBS in DMEM with 1% FBS. Cells were washed to remove unbound CPP-Nbs or Nbs, fixed with 4% paraformaldehyde for 10 min. Internalized HA-tagged Nbs were labeled with monoclonal rabbit anti-HA (1:1,600 dilution in PGS), followed by AF555-conjugated goat anti-rabbit IgG (1:200 dilution in PGS) at room temperature for 1 h each. Cells were analyzed on an Attune NxT acoustic focusing cytometer (Life Technologies) using the YL1 channel (excitation, 561 nm; emission, 585 nm). Data were analyzed by FlowJo software (Becton Dickinson) to visualize differences in relative percentages of cell populations among groups.

Cells were pretreated with various compounds, digested in Accutase, and then washed with Tyrode’s solution supplemented with 20% FBS. After counting, cell membranes were damaged with SLO toxin (1 μg/ml, 10 min) at 37°C. His-tagged SLO (carrying a cysteine deletion that eliminates the need for thiol activation) (37 (link)) was provided by R. Tweeten (University of Oklahoma, Norman, OK) and purified as described previously (13 (link)). SLO-treated cells were stained with PI (2 μg/ml) (Thermo Fisher Scientific) for 5 min and analyzed by the Attune NxT Acoustic Focusing Cytometer (Life Technologies). Total cell events were measured with Vybrant DyeCycle Violet stain (Invitrogen). At least 10,000 cells were used for each experimental group. Data were analyzed in Attune NxT software.