GSM8508352: PDR_S11; Homo sapiens; RNA-Seq
Vitreous tissue samples were collected in vitrectomy bags during surgery and processed for cell isolation within two hours of resection, according to a previously published protocol (11) adapted for the specificities of diabetic vitreous. For half of the samples in the preliminary analysis (see Table 1) and for diabetic samples, 1 ml of RBC Lysis Buffer (Thermo Fisher Scientific) was added for erythrocyte lysis. Samples were stained for cluster of differentiation (CD) 45 (BV421, anti-human, 1:100, BioLegend), CD11b (FITC, anti-human, 1:100, BioLegend), CX3C motif chemokine receptor 1 (CX3CR1, PE-Cy7, anti-human, 1:200, BioLegend) and Anti-Human Mature Macrophages (MatMac) Antibody, an ED2-like (ectodermal dysplasia 2) marker for resident macrophages (eFluor660, anti-human, 1:100, eBioscience). Finally, cells were processed for sorting on the MoFlo Asrios EQ Cytometer (Beckman Coulter). Hyalocytes were isolated as CD45+CD11b+CX3CR1+MatMac+ cells. RNA extraction, library preparation and RNA-Seq were conducted at the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Germany), according to previously published protocols (11,16).