Beckman type 50.2 ti rotor

Isolation and characterization of EVs was in accordance with the minimum information for studies of extracellular vesicles (MISEV) published in 2018 [42 (link)], as summarized by ISEV. EVs are characterized by their expression of vesicle-associated proteins (CD63, CD81, ITGAV) and diameter range of 40–150 nm. In this study, stromal cells (HCKs, HCFs, or HCMs) were cultured for 36 h in serum-free media, the conditioned media (CM) was collected, and EVs were isolated, as previously described [11 (link)]. In brief, stromal cell CM underwent serial differential centrifugation to remove cells (300× g for 10 min), cellular debris (3000× g, for 10 min), and apoptotic detritus (13,000× g for 30 min). The supernatant was concentrated using a Centricon^® Plus-70 centrifugal filter unit with a 100 kDa MW cutoff (MilliporeSigma, Burlington, MA, USA), and ultracentrifuged for 1 h and 10 min at 110,000× g (4 °C) using a Beckman Type 50.2 Ti Rotor (Beckman Coulter, Brea, CA, USA) in a Beckman Coulter, Optima LE-80K Ultracentrifuge. The resultant pellet was resuspended in phosphate buffered saline (PBS; Gibco), centrifuged again for 1 h and 10 min at 110,000× g (4 °C), and stored at −80 °C.

Isolated EVs were fluorescently labeled with a PKH26 Red Fluorescent Cell Linker Kit (MilliporeSigma) according to the manufacturer’s instruction [11 (link)]. The EV pellet was resuspended in diluent C, incubated with PKH26 dye in diluent C buffer at a ratio of 1:1 for 2 min at RT, and mixed with bovine serum albumin (BSA, 1% w/v in diluent C; Sigma Aldrich, St Louis, MO, USA) at an equal ratio per volume. The PKH26–EV solution was subjected to ultracentrifugation using a Beckman Type 50.2 Ti Rotor (Beckman Coulter) in an Optima LE-80K Ultracentrifuge (Beckman Coulter) at 110,000× g for 1 h and 10 min at 4 °C. The supernatant was removed and the PKH26-labeled EVs were washed, resuspended in diluent C, and ultracentrifuged (110,000× g for 1 h and 10 min at 4 °C). The wash/ultracentrifugation steps were repeated a total of three times. PKH26-labeled EVs were filter-sterilized (0.22 μm pore) prior to use in cell culture. For the control sample, particle-free PBS (Gibco) was used instead of EVs and stained according to the procedures described above.

EVs were isolated using standard ultracentrifugation step gradients based on published protocols [27 (link),28 ]. Briefly, hCE-TJ-conditioned medium or complete corneal fibroblast medium for hCE-TJ-EV or FBS-EV isolation, respectively, was collected on ice and subjected to successive centrifugation steps using a Beckman Type 50.2 Ti Rotor (Beckman Coulter, Brea, CA, USA) in an ultracentrifuge (Beckman Coulter, Optima LE-80K Ultracentrifuge) at increasing speeds (300× g, 10 min; 2000× g, 10 min; 11,004× g, 30 min; 146,000× g, 70 min) at 4 °C. The resulting pellet was re-suspended in the commercial total exosome isolation reagent (Invitrogen, Carlsbad, CA, USA) and phosphate-buffered saline (PBS) in a 1:2 ratio and incubated overnight at 4 °C with rocking, followed by centrifugation at 10,000× g for 60 min at 4 °C (Eppendorf Centrifuge 5417R, rotor F45-30-11, Hamburg, Germany). The isolated EV pellet was stored at −20 °C until further use.