All animal experiments were conducted after approval of the University of Cincinnati (UC) Institutional Animal Care and Use Committee (IACUC). The 1 × 104 MOLM-13 cells that express luciferase [21 (link)] were injected into 8-week-old male NCG mice (Charles River Laboratories, Wilmington, MA, USA) via the tail vein. On day 3 post-engraftment, mice were randomized to receive either vehicle (0.5% methylcellulose, 0.1% Tween80, 99.4% deionized water) or 82 mg/kg EPZ daily oral gavage. On day 10 post-treatment, mice in the EPZ cohort had their dose increased to 300 mg/kg [19 (link),22 (link)]. Early removal criteria (ERC) were defined as 20% weight loss, partial or full hindlimb paralysis, dehydration, anorexia, anemia, hunched posture, inactivity, lethargy, difficulty breathing, or rough hair coat.
Ncg mice
Manufactured by Charles River Laboratories
Sourced in United States
NCG mice are a genetically engineered mouse model developed by Charles River Laboratories. They are characterized by severe immunodeficiency, lack of T cells, B cells, and functional natural killer cells.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Biotium (2 mentions)
Fluoromount g mounting media, by Southern Biotech (2 mentions)
Tcs sp5 dmi inverted microscope, by Leica (2 mentions)
Fvmpe rs multiphoton microscope, by Olympus (2 mentions)
Nsg mice, by Jackson ImmunoResearch (1 mentions)
Ivis spectrum ct imaging system, by PerkinElmer (1 mentions)
14 protocols using ncg mice
1
Evaluating EPZ treatment in leukemia mice
2
Evaluating ARQ 531 and Venetoclax in MOLM-13 Xenografts
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A total of 10,000 luciferase expressing MOLM-13 cells [38 (link)] were engrafted via tail vein injection in NSG mice (The Jackson Laboratory, Bar Harbor, ME) for the single agent ARQ 531 study or NCG mice (Charles River Laboratories, Wilmington, MA) for the ARQ 531 + venetoclax (ARQ + Ven) combination study. For the first single agent ARQ 531 study, mice were treated 7 days post-engraftment. Due to the aggressiveness of the MOLM-13 model, mice were treated 4 days post-engraftment in the ARQ + Ven combination study. In both studies, mice were treated daily via oral gavage with vehicle (10% absolute alcohol, 10% Cremophor-EL, 80% saline) or 50 mg/kg ARQ 531, and 75 mg/kg venetoclax ± 50 mg/kg ARQ 531 for the combination study. Weekly in vivo imaging was performed using In Vivo Imaging System (IVIS) imager (PerkinElmer, Waltham, MA).
3
Humanized Keloid Xenograft Mouse Model
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Briefly, immediately after surgical excision, the human KS mass was washed twice with phosphate-buffered saline and cut into 4-mm square pieces under sterile conditions. The mice were anaesthetized by intraperitoneal injection of 5% chloral hydrate with 0.1 ml/10 g body weight, and 1-cm incisions were made on both sides of the dorsal midline above the gluteus maximus. The human keloid tissues were implanted into the subcutaneous pocket between the panniculus carnosus and skin, and the wound was sutured.
Eight-week-old NCG mice were purchased from Charles River (Beijing, China) and bred in the specific pathogen–free (SPF) murine facility. The First Hospital of Jilin University Research Animal Care Committee (Changchun, Jilin) approved the entire process, and all the animals were kept under standard conditions described in the guidelines approved by the institution.
Eight-week-old NCG mice were purchased from Charles River (Beijing, China) and bred in the specific pathogen–free (SPF) murine facility. The First Hospital of Jilin University Research Animal Care Committee (Changchun, Jilin) approved the entire process, and all the animals were kept under standard conditions described in the guidelines approved by the institution.
4
Xenograft Modeling of KRAS-Driven Tumors
5
Dual Xenograft Models for PET Imaging
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Two murine models were studied: a dual HER2+/HER2− flank model (n = 7) and a dual EGFRvIII+/EGFRvIII− flank model (n = 4). For the HER2+/HER2–model, 1 million MD468 (HER2+ breast cancer cell line, fast-growing) and 3 million SKBR3 (HER2− breast cancer cell line, slow-growing) subcutaneously injected xenografts generated roughly similar-sized tumors in 3 wk. Therefore, 4 × 106 SKBR3 cells and 1 × 106 MD468 cells were implanted subcutaneously into 6- to 10-wk-old female NCG mice (Charles River). For the EGFRvIII+/EGFRvIII– model, 1 × 106 EGFRvIII+ or EGFRvIII– U87 cells were implanted subcutaneously into 6- to 10-wk-old female NCG mice. All mice were then injected with 6.0 × 106 SNIPR T cells via the tail vein in 100 μL of PBS. For PET imaging, 5,550 MBq (150 mCi) of [18F]FHBG were administered via the tail vein. One hour after injection, images were acquired at days 3, 6, 8, and 10 after T-cell injection. On completion of imaging at day 10, the mice were killed, and biodistribution analysis was performed. Harvested tissues were γ-counted using a Hidex automatic γ-counter.
6
Immunohistochemical Staining of Tumor Tissues
7
Xenograft and Tail-Vein Injection Protocols
8
Xenograft Model for PIM Inhibitor Efficacy
9
Xenograft Modeling of KRAS-Driven Tumors
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SUIT2 and A549 xenografts were established in 4–8-week-old male NCG mice (Charles River, Wilmington, MA, USA). Mice were injected with 0.5×106KRAS4A−/− or KRAS4A+/+ cells into their contralateral flanks, and xenografts were grown for at most 6 weeks. Tumors were measured every 3–4 d with calipers. When tumors reached approximated 1 cm along their longest axis the mice were analyzed for glucose uptake by 18FDG PET/CT imaging. After imaging, the mice were sacrificed and tumors were excised, weighed and fixed in formalin. All animal protocols were approved by the NYU School of Medicine Institutional Animal Care and Use Committee (IACUC). Tumors were generated in mice under IACUC protocol IA16–00051, which allows tumors to grow to 2.5 cm3 or 2 cm in diameter. In no mouse did the tumor size exceed that allowable by the protocol.
10
Immunohistochemical Staining of Tumor Tissues
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All tissues were obtained from NOD/SCID/gamma mice (NCG mice, Strain Code: 572, Charles River Labs), fixed overnight in 10% neutral-buffered formalin, and embedded in paraffin. 5 μm serial sections of primary tumors and lungs were used for staining.
5 μm sections of paraffin-embedded tissues were deparaffinized in xylene, dehydrated with a graded series of alcohol, and rehydrated in 1X PBS. Heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) in a pressure cooker. After 2–3 h, sections were blocked with 10% donkey serum in 0.5% Tween 20 in 1X PBS for 2 h at room temperature, incubated with primary antibodies in 1% donkey serum in 0.5% Tween 20 in 1X PBS overnight at 4°C, and washed 3 times in 0.5% Tween 20 in 1X PBS. Fluorescently-labeled secondary antibodies (AlexaFluor, Molecular Probes) were added for 2 h at room temperature, and 10 μg/mL DAPI (Biotium) was added at the last wash to label DNA. Coverslips were mounted with Fluoromount-G Mounting Media (Southern Biotech). A Leica TCS SP5 DMI inverted microscope was used for fluorescent imaging of tissue sections (40× NA 1.25, 63X, NA 1.4).
5 μm sections of paraffin-embedded tissues were deparaffinized in xylene, dehydrated with a graded series of alcohol, and rehydrated in 1X PBS. Heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) in a pressure cooker. After 2–3 h, sections were blocked with 10% donkey serum in 0.5% Tween 20 in 1X PBS for 2 h at room temperature, incubated with primary antibodies in 1% donkey serum in 0.5% Tween 20 in 1X PBS overnight at 4°C, and washed 3 times in 0.5% Tween 20 in 1X PBS. Fluorescently-labeled secondary antibodies (AlexaFluor, Molecular Probes) were added for 2 h at room temperature, and 10 μg/mL DAPI (Biotium) was added at the last wash to label DNA. Coverslips were mounted with Fluoromount-G Mounting Media (Southern Biotech). A Leica TCS SP5 DMI inverted microscope was used for fluorescent imaging of tissue sections (40× NA 1.25, 63X, NA 1.4).
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