Ml microcentrifuge tube

For steroids quantification by LC-MS/MS analysis, 200 µL of DPS containing IS was added into each 2.0 mL microcentrifuge tube (Eppendorf^®, Hamburg, Germany). Subsequently, 100 µL of serum sample from each patient, calibrators, and QCs was added into a tube with DPS, mixed (20 °C, 15 min) in a Thermomixer (Eppendorf^®), and then centrifuged (4210 rcf, 20 °C, 30 min). The organic layer (175 µL) was recovered, dried in a SpeedVac, and reconstituted with 125 µL of H₂O:MeOH 60:40. The obtained solution was gently mixed in a Thermomixer (20 °C, 15 min) and briefly centrifuged, and the supernatant was transferred into a polypropylene vial (Waters Corporation, Milford, MA, USA). The vials were placed in the autosampler for LC-MS/MS analysis. The LC-MS/MS system consisted of a HPLC Alliance HT 2795 Separations Module coupled to a Quattro UltimaPtESI tandem quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA), operating in positive electrospray ionization mode with MassLynx v4.1 software (Waters). An amount of 50 µL was injected, with a total run time of 18.00 min. A detailed description of the LC-MS/MS analysis for the determination of steroids and the instruments parameters have already been reported [21 (link)]. QuanLynx 4.1 software (Waters Corporation, Milford, MA, USA) was used for data processing and quantification.

Tear Schirmer’s strip samples were cut into 2–3 mm paper pieces and transferred into a 0.5 mL microcentrifuge tube (Eppendorf®, Hamburg, Germany), paying attention to wash the required equipment with EtOH before and after each sample preparation. After adding 200 µL of 0.01M PBS, Phosphate Buffered Saline (Sigma-Aldrich, St. Louis, MI, USA), each sample was gently mixed (22 °C, 10 min, 700 rpm) in a Thermomixer (Eppendorf®, Hamburg, Germany). After, the 0.5 mL tubes containing the piece of the strip in PBS were cut off on the bottom, put into another 2.0 mL microcentrifuge tube (Eppendorf®, Hamburg, Germany) and centrifuged (room temperature, 5 min, 13000 rpm). The extracted liquid (195 µL) was transferred into 1.5 mL microcentrifuge tube (Eppendorf®, Hamburg, Germany), aliquoted and used for subsequent FC analyses in order to sort pure EVs or for proteomics investigation.
Proteomics analyses were executed on whole lacrimal fluid from 5 subjects belonging to the two different clinical groups. Tear proteins were first extracted, pooled and then quantified by Bradford assay (Bio-Rad, Hercules, CA, USA), using Bovine Serum Albumin (BSA, Sigma-Aldrich, St. Louis, MI, USA) standard for the calibration curve. 50 µg of proteins was digested for each clinical group.