Redifect red fluc gfp

Patient-derived GBM BTIC line GBM1A, originally established as line 020913 [40 (link)] and extensively characterized by our collaborators, was cultured as neurospheres in Dulbecco's modified Eagle's medium/F-12 medium supplemented with EGF and FGF (20 ng/mL each). To localize cells in vivo, cells were transduced with a lentivirus for GFP-luciferase (GFP-luc; RediFect™ Red-FLuc-GFP, Perkin Elmer CLS960003) and sorted using fluorescence-activated cell sorting for GFP.

We utilized a primary cell line of human GBM cells (GBM1A, also known as line 020913) (27 (link)). GBM1A cells were transduced with a GFP-luciferase lentivirus (RediFect™ Red-FLuc-GFP, Perkin Elmer CLS960003) and sorted for GFP positivity. Following cell transduction, intracranial implantation of tumors was performed as previously described (28 (link)–31 (link)). Briefly, mice were anesthetized and placed in a stereotactic frame. 5.0 x10⁵ GBM1A-GFP luciferase+ cells were injected in 2 μL of DMEM/F12 into the right brain hemisphere. 3 injection sites were established in the following coordinates (in mm relative to bregma); LV-proximal: AP: 1.0, L: 1.2, D: 2.3, n = 17; LV-intermediate: AP: 1.5, L: 1.3, D: 3, n = 7; and LV-distal: AP: 1.0, L: 2.1, D: 2.3, n = 17. Tumor growth was monitored weekly by bioluminescence following luciferin injection. For survival experiments, mice were maintained until reaching humane endpoint criteria following GBM xenograft. For histology analysis, mice were maintained for 4 weeks after tumor implantation (n=7). Mice were then anesthetized and perfused with 4% paraformaldehyde. Brains were extracted and cryoprotected in 30% sucrose. Brains were sectioned using an HM 430 Freezing Microtome at 30 μm thickness. Sections were stored in 30% ethylene glycol, 20% glycerol, 0.05M PBS, pH 7.4 at -20°C until immunohistochemical processing.

The 4T1-BR5 cells were received from Dr. Patricia Steeg’s lab and engineered to stably co-express red-shifted Luciola Italica luciferase (Red-FLuc) and green fluorescent protein (GFP) using a commercial lentiviral vector (RediFect Red-FLuc-GFP; PerkinElmer, USA). Cells were transduced and FACS sorted based on GFP expression using a FACSAria III flow cytometric cell sorter (BD Biosciences). The resultant 4T1BR5-Red-FLuc/GFP cells were maintained in DMEM containing 10% FBS and 1% antibiotics, at 37 °C and 5% CO₂. For iron labeling, 2 × 10⁶ 4T1BR5-Red-FLuc/GFP cells were plated, and 24 hours later were incubated with 25 μg/mL of micron-sized superparamagnetic iron oxide (MPIO) beads for an additional 24 hours (0.9 μm in diameter, 63% magnetite, conjugated with Flash Red; Bangs Laboratory, Fishers, IN, USA). Cells were washed three times with Hanks balanced salt solution (HBSS), collected and thoroughly washed three more times with HBSS to wash off residual unincorporated MPIO before in vitro evaluation or injection into animals.

The 4T1BR5 cells were a kind gift from Dr. Patricia Steeg's lab and transduced with a commercial lentiviral vector (RediFect Red-FLuc-GFP; PerkinElmer, USA). Cells were FACS sorted based on GFP expression using a FACSAria III flow cytometric cell sorter (BD Biosciences, USA). The parental 4T1 cells were also received from Dr. Patricia Steeg's lab and transduced with an in-house RLuc8/ZsG lentivirus. Cells were sorted based on ZsG expression using FACS. The resultant 4T1BR5-FLuc/GFP (4T1BR5-Fluc) and 4T1-RLuc/ZsG (4T1-Rluc) cells were maintained in DMEM containing 10% FBS and 1% antibiotics, at 37°C and 5% CO₂, and then transduced a second time using an in-house CD:UPRT/tdT lentivirus. Both cell lines were FACS sorted based on tdT expression. All lentiviral transductions were performed using a multiplicity of infection of 20 and in the presence of 8 μg/mL of polybrene. Cells were washed three times with Hanks balanced salt solution (HBSS) and collected for in vitro evaluation or injection into animals.