Softmax pro software 5.4

CD20 was measured on the surface of serum derived EVs using 96-well ELISA plates coated with Human B-lymphocyte MS4A1 (CD20) (Catalog # MBS283766; MyBioSource, Inc. San Diego, CA, USA). Briefly, EV samples were diluted at 1:2 in 1X PBS containing 0.1% Tween 20 before loading onto ELISA plates and the assay was carried out according to manufacturer’s instructions (MyBioSource, Inc.). Results were acquired for CD20 standards and EV samples from two independent reproducible readings of duplicate wells at 450 nm using a VersaMax Absorbance Microplate Reader (Molecular Devices, LCC., San Jose, CA, USA) with SoftMax Pro Software (5.4) for Data Acquisition & Analysis.

IGF-1 and IGFBP-3 levels were measured from T1DM and HC serum samples that were obtained from the MERIT study using the Human IGF-I/IGF-1 Quantikine^® ELISA Kit (R & D Systems, Minneapolis, MN, USA) and Human IGFBP-3 Quantikine^® ELISA Kit (R & D Systems), respectively, according to the protocol set by the manufacturer. Serum samples underwent a 100-fold dilution in Calibrator Diluent RD5-18. SpectraMax^® 190 absorbance plate reader (Molecular Devices, San Jose, CA, USA) and SoftMax^® Pro Software 5.4 (Molecular Devices) were used to analyze the data from both the IGF-1 and the IGFBP-3 assays. A standard curve was created by generating a log/log curve fit. Both the IGF-1 and the IGFBP-3 assays were read at 450 nm with correction at 570 nm.