Pluronic f 127

THP-1, Ramos, and Jurkat cells
were loaded with Fura-2-AM (4 μM, Abcam) in HBSS assay buffer
[1× HBSS (Corning), 10 mM HEPES (pH 7.4), 1.8 mM CaCl₂, 0.8 mM MgCl₂, and 0.1% BSA] containing 0.04% Pluronic
F127 (Thermo Fischer Scientific) at 37 °C for 40 min and at RT
for additional 20 min. Fluorescence was evoked by 340 and 380 nm excitation
wavelengths and emission was read at 510 nm using a fluorescence plate
reader (Tecan2000). Data were presented as 340/380 fluorescence ratios
representative of changes in the intracellular Ca²⁺ level.

After transfection for 40 h, primary CD3⁺ T cells were stimulated using Dynabeads™ Human T Activator CD3/CD28 (bead to cell ratio, 1:4; Thermo Fisher). Proliferation and apoptosis were carried out according to previously described methods (49 (link)). In order to detect calcium (Ca²⁺) flux, CD3⁺ T cells were stained with LIVE/DEAD™ dye (Thermo Fisher), followed by loading with Fluo-4-AM, Fura-red, and Pluronic F-127 (Thermo Fisher) for 30 min. Cells were then labeled with antibodies against CD3, CD4, and CD8 for 20 min. To record Ca²⁺ flux, a baseline fluorescence signal was acquired for 90 s. Following this period, the cells were stimulated with soluble anti-CD3/CD28 (10 μg/mL) (50 (link)), after which fluorescence signal recording was continued for 300 s. Ionomycin (1 μg/mL) was used to elicit a maximum response for another 120 s. For quantification of Ca²⁺ flux, the ratio of Fluo-4 or Fura-red at the peak of the response relative to the baseline was determined (51 (link)). Cells were detected using the LSR II flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo software (Ashland, OR, USA).

Isolated EVs were subjected to vesicle flow cytometry (vFC) using a specific fluorescent membrane dye Di8-ANEPPS (D3167, Thermo-Fisher) as previously described30. Briefly, EV samples were labeled with Di8-ANEPPS staining buffer (12 μM) supplemented with Pluronic F-127(#P6866, Thermo-Fisher) at room temperature for 30 min and then stained with following APC-conjugated antibodies listed in Table 1. Configuration of the MACSQuant10 (MQ10) was set to trigger on events over 1.1 V on the basis of Di8-ANEPPS (B3 channel on the MQ10 to focus analysis on dye-labeled events. Additional details are provided in the Supplementary information. To identify detergent soluble EVs and to avoid ‘swarm’ and other background artifacts, samples were analyzed twice, first without detergent and then second after the additive of the detergent Triton X-100 (T8532, Sigma Aldrich, St. Louis, MO, USA). This sequential strategy enables the selection of a gate that identifies detergent soluble Di8-ANEPPS positive EVs from which additional staining with high index fluorophore such as APC can be used for the detection of antibodies bound to EVs.

Fully differentiated iWAT cells with the indicated genotype were seeded at 40,000 cells per well in collagen I-coated 96-well black clear-bottom plates and incubated overnight. Intracellular calcium concentration ([Ca]_i) was monitored with the [Ca]_i indicator Fluo-4 AM (F14201, ThermoFisher). Cells were loaded with 5 μM Fluo-4 AM and 0.02% Pluronic F-127 (P3000MP, ThermoFisher) for 30 min at 37 °C in Hank's balanced salt solution (HBSS). Cells were then washed twice and incubated in HBSS for 30 min at room temperature. Before fluorescence recording, the buffer was changed with calcium-free HBSS. Following baseline recording (F₀), 2 μM thapsigargin or 50 μM 2-APB was added at the indicated time-point and then the fluorescence was recorded. Fluorescence signals were measured with a Spectramax M5 multimode plate reader (Molecular Devices; excitation, 494 nm; emission, 516 nm).

Magnesium Green-AM (M3735; Thermo Fisher Scientific) was applied to the DMEM culture medium at 10 µg/ml with 0.02% Pluronic F-127 (P3000MP; Thermo Fisher Scientific), and the cells were incubated at 37°C for 1 h. The cells were then washed twice with HBSS (14170112; Thermo Fisher Scientific) and further incubated in fresh HBSS at 37°C for 15 min to complete hydrolysis of the acetoxymethyl ester form. Magnesium Green fluorescence was measured by DeltaVision equipped with an Olympus PlanApoN 60× objective (NA 1.42) and an sCMOS camera with an FITC filter by using Softworx software. The cell culture conditions (37°C, 5% CO₂, and humidity) were maintained in a live-cell chamber under the microscope. The nucleoplasm intensity was measured by using Softworx software after subtraction of background signals outside cells and plotted.