Eight-week-old male nude mice (Orient Bio) weighing approximately 25 g were used for the experiment to prevent the rejection of human fat tissue grafts. The control and the test groups were placed on the backs of the same mouse to minimize variance between the experimental results. The isolated SVF and the plasminogen peptides (PLP-1 and PLP-3) were added to the adipose tissue. Then, 20 μL of SVF and peptides were added to 80 μL of PBS. One hundred microlitres of the test sample (SVF and peptide mixture) were mixed with 300 μL of human fat tissue. After gentle mixing, the fat with the test sample was transferred into 1-mL Luer-Lock syringes for placement of the graft. Each mixture (total volume 0.4 mL) was injected using an 18-gauge needle. Six mice were sacrificed four weeks after transplantation, and the grafted tissues were harvested.
Nude mice
Manufactured by Orient Bio
Nude mice are a type of laboratory animal that lack a functional immune system. They are commonly used in research to study the development and progression of various diseases, as well as the efficacy of new therapeutic treatments. Nude mice are characterized by their lack of fur and the absence of a functional thymus gland, which is responsible for the development of T cells, a critical component of the immune system.
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5 protocols using nude mice
1
Enhancing Fat Tissue Grafting in Mice
2
SK-BR-3 Xenograft Tumor Model and Treatment
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Nude mice at 4–5 weeks of age were purchased from Orient Bio. For a SK-BR-3 xenograft model, 20 million of SK-BR-3 cells were xenografted at the right flank of the mice with matrigel (Corning). After the volume of SK-BR-3 tumor reaches approximately 130 mm3, 6 µg of AlBD-SC-RTA-KDEL, AlBD/HER2Afb/RTA, or AlBD/HER2Afb/RTA-KDEL were administrated intravenously for each group (100 µl each) for 8 times in 2 or 3-day intervals. Tumor volumes and body weights were measured with a caliper and weighing scale, respectively, and actual final weights of tumor masses were assessed by biopsy at day 47. Major organs (liver, kidney, spleen, heart, and lung) were also biopsied simultaneously and fixed with 10% formalin (Sigma-Aldrich). The biopsied and fixed organs and tumor masses were made into paraffin blocks, sectioned, and stained with hematoxylin and eosin (H&E). DNA fragmentation in the paraffin section of the tumor masses were stained by TUNEL assay, to monitor apoptosis occurred in the tumors. H&E staining and TUNEL assays was done with the aid of LABCORE, Inc.
3
In Vivo Xenograft Study of Ceritinib
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Nude mice (Orient Bio, Sungnam, Korea) were used for in vivo studies and were cared for in accordance with the guidelines approved by the Samsung Biomedical Research Institute (protocol No. H-A9–003) and the Institute of Laboratory Animal Resources Guide. 8-week-old female mice were injected subcutaneously with 5 million A375P cells together with Matrigel. Once tumors reached an average volume of 100 mm3, mice were randomized to the different treatment cohorts; that is, they were randomized to receive ceritinib (20 or 50 mg/kg body weight per os (p.o.) daily for 17 days) or vehicle control (n = 5 for LDK-378, 20 mg or 50 mg; n = 5 for vehicle control). Mice were observed daily throughout the treatment period for signs of morbidity and/or mortality. Tumors were measured twice weekly using calipers, and tumor volume was calculated using the formula: length × width2 × 0.52. Body weight was also assessed twice weekly. The p values were determined with the Wilcoxon rank-sum test.
4
Nude Mouse Model for Research
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Twenty four-week old nude mice (weight: about 15∼20 g) from Orient Bio Inc. (Seongnam, Korea) were used for the animal model portion of this study. After a five day adjustment period, we checked that the mice were normal, then began the experiment. The animals were freely fed with hard food and water at 22°C temperature and 12 hours light contrast cycle. We observed the policy of Korean Association for Laboratory Animal Science and regulations related to animal experiment.
5
Evaluating the Therapeutic Potential of SNHG15 Knockdown in Osteosarcoma
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Nude mice were obtained from Orient Bio Inc (Seongnam, South Korea). The stable SNHG15-knockdown MG63/DXR cells were built by transfection with short hairpin RNA (shRNA) against SNHG15 (sh-SNHG15; Santa Cruz Biotechnology, Dallas, TX, USA), and MG63/DXR cells stably transfected with sh-NC were used as the control. The mice (n = 5 in each group) were subcutaneously injected with the abovementioned MG63/DXR cells stably transfected with sh-NC or sh-SNHG15. DXR (5.0 mg/kg) and PBS were injected into mice through the tail vein every 2 days since the average tumor size reached about 50 mm3. The tumor volume was recorded every 4 days and calculated using the formula: width2 × length × 0.5. The mice were sacrificed after 31 days of inoculation, and the tumors were dissected to measure weight and detect the levels of SNHG15, miR-381-3p, and GFRA1.
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