We used male athymic nude mice (6 wk old; Central Lab. Animal Inc.). The mice were housed in micro-isolator cages under sterile conditions and observed for at least 1 week before study initiation, to ensure proper health. Lighting, temperature, and humidity were centrally controlled. Dissociated GBM TSs (5 × 105 per mouse) were implanted into the right frontal lobe of mice at a depth of 4.5 mm, using guide-screw system [35 (link)]. The mice were randomly allocated based on their body weights without blinding (n = 5 mice per group). If the body weight decreased by more than 15% relative to the maximum weight, mice were euthanized according to the approved protocol. For bioluminescence acquisition and analyses, mice were injected intraperitoneally with 100 μL D-luciferin (30 mg/mL; Promega) under 2.5% isoflurane anesthesia, 15 min before signal acquisition, and then observed using IVIS imaging system and Living Image v4.2 software (Caliper Life Sciences). For immunohistochemistry, sections (5 μm thick) were obtained using a microtome and transferred onto adhesive slides. Antigen retrieval and antibody attachment were performed using the Discovery XT platform (Ventana Medical Systems). Zeb1 was detected using a peroxidase/DAB staining.
Male athymic nude mice
Manufactured by Central Lab Animal
Male athymic nude mice are laboratory animals with a genetic mutation that results in a lack of thymus gland development and a weakened immune system. They are commonly used in medical research, particularly for the study of human cancers and the evaluation of new treatments.
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6 protocols using male athymic nude mice
1
Glioblastoma Xenograft Model in Mice
2
Orthotopic Glioblastoma Xenograft Model
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Male athymic nude mice (Central Lab Animal Inc., Seoul, Korea), aged 6 to 8 weeks, were used in this study. U87MG GBM cells were implanted into the right frontal lobe of nude mice using a guide-screw system within the skull, as described previously [48 (link),49 (link)]. A total of 2 × 105 U87MG GBM cells were injected into each mouse (sh-Ctrl (n = 5); sh-Ctrl+IR (n = 4); sh-CD44+IR (n = 4)) using a multiple micro-infusion syringe pump (Harvard Apparatus, Holliston, MA, USA) at a speed of 0.5 μL/min. Mice with a similar weight and age were randomized to the experimental groups.
3
Athymic Nude Mouse Study Protocol
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Male athymic nude mice (Central Lab Animal Inc., Seoul, Korea), aged 4–8 weeks, were used in this study. Mice were housed in micro-isolator cages under sterile conditions and were observed for at least 1 week before study initiation to ensure proper health. Lighting, temperature, and humidity were centrally controlled. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Yonsei University College of Medicine.
4
Modeling Glioblastoma in Athymic Nude Mice
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Male athymic nude mice (6 weeks old; Central Lab. Animal Inc., Seoul, Korea) were used in this study. Mice were housed in micro-isolator cages under sterile conditions, and observed for at least 1 week before the study initiation to ensure proper health. Lighting, temperature, and humidity were controlled centrally. Dissociated GBM TSs (5×105 cells/mice; usual cell number for GBM patient-derived TSs) and the same number of tMSLCs or vMSLCs were implanted into the right frontal lobe of mice at a depth of 4.5 mm using the guide-screw system. If body weight decreased by more than 15% compared to the maximum, mice were euthanized according to the approved protocol. For immunohistochemistry, 5-µm-thick sections were obtained using a microtome and transferred onto adhesive slides. Antigen retrieval and antibody attachment were performed using an automated instrument (Discovery XT). Zeb1 was detected using a peroxidase/DAB staining system. All experimental procedures involving animals were approved by the Yonsei University College of Medicine Institutional Animal Care and Use Committee (2017-0347). Informed consent was obtained from all individual participants included in the study.
5
Glioblastoma Xenograft Mouse Model
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Male athymic nude mice (4–8 weeks old; Central Lab. Animal Inc., Seoul, Korea) were used in this study. Mice were housed in micro-isolator cages under sterile conditions and monitored for at least 1 week before study initiation to ensure proper health. Lighting, temperature, and humidity were controlled centrally. Mice were anesthetized with a solution of Zoletil (30 mg/kg; Virbac Korea, Seoul, Korea) and xylazine (10 mg/kg; Bayer Korea, Seoul, Korea), which was administered intraperitoneally. GBM TSs (GSC11) were pretreated by niclosamide (500 nM), TMZ (250 µM), and combination of niclosamide and TMZ for 3 days with reference to the pretreatment method from several studies (Liu et al. 2016 (link); Natale et al. 2018 (link); Wang et al. 2019 (link); Xia et al. 2017 (link)). DMSO-control (n = 5), niclosamide (n = 5), TMZ (n = 5), and combination of niclosamide and TMZ (n = 5)—pretreated GBM TSs were implanted into the right frontal lobe of nude mice using a guide-screw system (Lal et al. 2000 (link)). total of 5 × 105 cells was injected to a depth of 4.5 mm using a Hamilton syringe (Dongwoo Science Co., Seoul, Korea). Mice were euthanized according to the approved protocol if daily monitored body weight had decreased by more than 15% compared to the original body weight.
6
Intracranial Xenograft Model in Athymic Mice
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Male athymic nude mice aged 4-8 weeks (Central Lab. Animal Inc.,Seoul, Korea) were housed in microisolator cages under sterile conditions and monitored for at least one week before study initiation to ensure proper health. Lighting, temperature, and humidity were controlled centrally. Dissociated Luc-TS13-64 cells were pretreated with DFS for 72 h using the pretreatment protocol [27, 28] . Cells were selected by trypan blue staining, and 5×10 5 viable cells were implanted into the right frontal lobe of each mouse at a depth of 4.5 mm using a guide-screw system [21] [22] [23] 29] . Mice showing a >15% reduction in body weight compared with the maximum were euthanized according to the approved protocol.
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