Ab38496

Approximately 2×10⁶ ethanol-fixed PBMCs from two patients prepared during the previous studies [15 (link), 21 (link)] were washed twice in 40 mL ice-cold 1× PBS/1% fetal calf serum (FCS), collected via centrifugation (1,500 rpm for 5 min), and re-suspended in 500 mL cold 1× PBS/0.5% bovine serum albumin (BSA) buffer. Approximately 100 mL of this cell suspension was incubated with the following antibody combinations for 30 min at 4°C in the dark: MAGEC1- Alexa488/CD34-PE/CD45-PerCP and MAGEA3-Alexa647 or PRAME-Alexa647 and MAGEA3-Alexa647/PRAME-Alexa488/ CD34-PE/CD45-PerCP; MAGEC1-Alexa488/CD138-PE/CD45PerCP and MAGEA3-Alexa647 or PRAME-Alexa647. All antibodies were used at a final concentration of 5 mg/mL. MAGEC1 (Abcam, clone ab115351), MAGEA3 (Abcam, clone ab38496) and PRAME (Abcam, clone ab135600) antibodies were fluorescently labeled with the Mix-n-Stain Alexa 647 kit (Sigma-Aldrich). The labeled cells were washed with 1 mL cold 1× PBS/0.5% BSA buffer at 1,500 rpm for 5 min; the pellet was re-suspended in 1 mL cold 1× PBS/0.5% BSA buffer and stored in the dark until analysis. The cells were analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using the Cell Quest Pro software, applying parameters and gates that had been previously optimized to minimize spectral overlap and increase sensitivity [15 (link), 21 (link)].

Printing of Abs to be used for exosome capture in microarrays was performed as described in Supplemental Methods. The following Abs were used for capture of total exosomes: anti-CD9 (ab58989, Abcam, Cambridge, UK), anti-CD63 (NBP2-42225AF647, Novusbio), anti-CD81 (ab59477, Abcam). The Abs cocktail for the capture of tumor-enriched exosomes (TEX) consisted of anti-EGFR1 (ab52894, Abcam), anti-MAGEA3 (ab38496, Abcam), anti-EpCAM (ab75813, Abcam) and anti-CSPG4 (a gift from Dr. Soldano Ferrone, Harvard Medical School). As positive and negative controls, human IgG and PBS were used. Figure 2(a) is a graphic illustrating the strategy used for capture of TEX from plasma-derived total exosomes. Titrations of optimal Ab concentrations for TEX capture and testing of microarrays specificity for TEX are illustrated in Figure 2(b) and described in Supplemental Methods.