TICs within the two passages subsequent to lentiviral transduction were harvested with accutase and resuspended in serum-free supplemented-medium. Three hundred thousand cells were injected into the pons of adult mice in a total volume of 3 μL (i.e. 100,000 cells/μl) as described above. Mice were kept under specific pathogen-free conditions in air-filtered cages and received food and water ad libitum. Tumor growth was monitored using an IVIS 50 system with charge-coupled device (CCD) camera (PerkinElmer) every 2-3 weeks. Mice were anesthetized as described previously and imaged 10 min after intra-peritoneal injection of luciferin (150 mg/kg body weight, Promega). Radiance signal intensity (normalized photon counts in photons/second/cm2/steradian) in the pons region was quantified using Living Image software (Perkin Elmer). Doubling time of the CDOX models was estimated based on bioluminescence exponential curves.
Ivis 50 system
Manufactured by PerkinElmer
Sourced in United States
The IVIS-50 system is a high-performance in vivo imaging platform designed for researchers. It provides non-invasive optical imaging capabilities to visualize and quantify various biological processes in small animals. The system utilizes bioluminescence and fluorescence imaging techniques to detect and monitor cellular and molecular events.
Automatically generated - may contain errors
4 protocols using ivis 50 system
1
Transplantation of Tumor Cells in Mice
2
Orthotopic Pancreatic Cancer Xenograft Model
3
GD2 CAR T-Cell Cytotoxicity Assay
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1 x 104 GL26 luc2-GFP/GD2 or SB28 luc2-GFP/GD2 tumor cells were plated per well in black 96-well plates. The following day, GD2 CAR T-cells or mock activated T-cells were added in each well at a specific effector to target ratios, ranging from 1:1 to 1:32 effector to target (312 to 1 × 104 T-cells). For cell killing assays, imaging of luciferase was performed after 24 hours of T-cell and tumor cell co-incubation on an IVIS-50 system (PerkinElmer, Waltham, MA) after addition of 56 μg D-luciferin (PerkinElmer) to each well. Images were taken with an exposure time of 30 s, f/stop of 1, and medium binning. For cytokine release assays, the supernatant was removed from individual wells and analyzed for IFN-γ, TNF-α, and IL-2 via an ELISA assay per the manufacturer instructions (Invitrogen, Carlsbad, CA).
4
Evaluating CAR T-Cell Cytotoxicity
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To evaluate the cytotoxicity of CAR or mock T cells as single agents, we plated 2 × 104 B16-TurboRFP/RLuc8 or B16-TurboRFP/RLuc8-mCD19 cells in 100 μL of DMEM in black 96-well plates. The following day, T cells were added at specified E:T ratios based on the initial number of plated tumor cells, and a viable fraction of tumors cells was measured 24 and 48 h thereafter by comparing fractional bioluminescence signal from RLuc8 between treated and untreated wells. Imaging of RLuc8 was performed on an IVIS-50 system (PerkinElmer, Waltham, MA, USA) immediately after washing cells once with PBS and addition of 200 μL of 1 μg/mL coelenterazine (NanoLight Technologies, Pinetop, AZ, USA) to each well. Images were taken with an exposure time of 60 s, F-stop of 8, and medium binning.
In combination studies with both VV and T cells, B16-TurboRFP/RLuc8 or SB28-TurboRFP/RLuc cells were instead plated at 104 cells/well (to account for longer duration of experiment) in 100 μL DMEM. The following day, the media were removed and replaced with 150 μL DMEM containing either Ctrl or mCD19 VV at an MOI of 0.2 (B16), 0.05 (SB28), or 0.1 (B16-mCD19low). After 48 h of infection, T cells were added at an E:T ratio of 4:1 (B16 and SB28) or 1:1 (B16-mCD19low) relative to initial number of plated cells (given the time delay from initial cell plating), and viability was assayed as previously described.
In combination studies with both VV and T cells, B16-TurboRFP/RLuc8 or SB28-TurboRFP/RLuc cells were instead plated at 104 cells/well (to account for longer duration of experiment) in 100 μL DMEM. The following day, the media were removed and replaced with 150 μL DMEM containing either Ctrl or mCD19 VV at an MOI of 0.2 (B16), 0.05 (SB28), or 0.1 (B16-mCD19low). After 48 h of infection, T cells were added at an E:T ratio of 4:1 (B16 and SB28) or 1:1 (B16-mCD19low) relative to initial number of plated cells (given the time delay from initial cell plating), and viability was assayed as previously described.
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