Cell trace carboxyfluorescein succinimidyl ester cfse

Dextran (50,000), Dextran sulfate (7000–20,000), chondroitin sulfate, heparin, N-acetyl heparin, and trypsin-EDTA (0.25%) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the powder reagents were dissolved in sterile water. FITC- or PE-labeled anti-CD138, anti-CD4, anti-CD19, and monensin were purchased from Biolegend (San Diego, CA, USA). FITC-labeled anti-CD56 and PE-Cy5-mouse anti-human CD107a were purchased from BD Pharmingen (San Diego, CA, USA). FITC-labeled anti-human or anti-mouse IgG Fc was purchased from Sigma (St. Louis, MO, USA). PE-conjugated streptavidin was purchased from BD Biosciences (San Jose, CA, USA). Cell trace carboxyfluorescein succinimidyl ester (CFSE) was purchased from Invitrogen (Carlsbad, CA, USA). Protein A/G agarose and heparin sepharose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and Abcam (Cambridge, UK), respectively.

To assess the proliferative response to a secondary allogenic stimulation, T-alloA210, T-allo10, and T-allo cells were labeled with CellTrace™ carboxyfluorescein succinimidyl ester (CFSE; Life Technologies, Carlsbad, CA, USA), and plated with allogenic mDC (from the same donor used during the primary stimulation) at a 10:1 T:DC ratio, in 200 µL of complete medium in 96-well round bottom plates. To test if T-alloA210 cells were able to respond to a polyclonal stimulation, the cells were stimulated with Dynabeads coated with anti-CD3 and anti-CD28 antibodies (Life Technologies, Carlsbad, CA, USA) at a 1:20 bead:cell ratio. After 72 h, the percentage of CFSE-low divided cells within the CD3⁺CD4⁺ population was assessed by flow cytometry. Percentage of T-cell anergy was calculated using the formula: “(% proliferation T-allo with mDC – % proliferation T-alloA210 or T-allo10 with mDC)/% proliferation T-allo with mDC.” Stability of T-alloA210 and T-allo10 cell anergy was tested in the presence or absence of 5 ng/mL of rhIL-1β, rhTNF-α, and rhIL-6 (R&D Systems, Minneapolis, MN, USA) upon 72 h stimulation by allogenic mDC.

EVs were re-suspended in 400 μL PBS at a concentration of 0.1–0.2 μg. The EVs were dyed by CellMask Deep Red (Thermo Fisher Scientific), with excitation/emission wavelength at 649/666 nm. EVs were stained with crimson dye (1:1000) at 37 °C for 20 min for labeling and washed with PBS (1 to 10,000 v/v ratio) to remove the unbound dye. Then, the EVs were separated by centrifugation at 100,000×g for 1 h and resuspended in PBS. Next, the EVs were stained with CellTrace™ carboxyfluorescein succinimidyl ester (CFSE, Life Technologies, Carlsbad, CA) and observed at 492/517 nm. The CFSE dyes were permeated into detached cells using endoesterase and subsequently reacted with intracellular primary amines to form a stable and non-diffusible fluorescent staining. LL29 cells (3–5 × 10⁵) in serum-free medium were dyed by CFSE at a 1:1000 dilution (5 μM) and incubated at 37 °C for 20 min under conditions devoid of light. The solution was then centrifuged and the pellet was rinsed with a serum-free medium at a ratio of 1:10 to eliminate the unbound dye.
EVs were then cultured in an 8-well chambered slide (Millipore, Burlington, MA). CFSE-dyed cells were treated with EVs, fixed with 3.7% (w/v) formaldehyde at ambient temperature for 5 min, and subsequently imaged with a fluorescence microscope.

Human peripheral blood red blood cells (RBCs) from healthy human volunteers were isolated by centrifugation and aspiration of the platelet-rich plasma and buffy coat layer. Erythrocytes were purified by resuspension in RPMI-1640 (Lonza, NJ) [10% hematocrit] and centrifugation (500 X g) followed by aspiration of the top 10% of the erythrocyte layer (this purification procedure was carried out with six repetitions) (24 (link)). Purified erythrocytes were then resuspended (20% hematocrit) at 4°C for ~24–36 h to induce apoptosis. Erythrocytes were next washed twice with DPBS (ThermoFisher Scientific, MA) and then stained with Cell Trace™ Carboxyfluorescein succinimidyl ester (CFSE) at a concentration of 5mM for 30 min at 37°C (ThermoFisher Scientific, MA).