Female 5-week-old NMRI-Foxn1 nu/nu mice (16–20 g) (Janvier Labs, France) were injected subcutaneously with 4 × 106 of SKMEL28 cells or, after Ketamine/Xylazine anesthesia, were xenografted with a patient-derived BRAF V600E-mutated tumor tissue in the brown fat. Mice were euthanized by cervical dislocation when their tumor reached a volume of approximately 1-1.5 cm3. Tumors were excised and a representative tumor piece was washed with PBS and sliced. Tumor sections were then placed in wells coated with gelatin (Sigma-Aldrich, USA) in 24-well plates in quadruplicates. The tumor sections were incubated for 8 days at 37 °C in 250 μL complete media supplemented with 10 μM dabrafenib (GSK2118436, Selleckchem), 1 μM cobimetinib (GDC-0973, RG7420, Selleckchem), or a combination of both inhibitors dissolved in DMSO. As controls, some wells received an equivalent volume of medium containing 0.5% DMSO. The medium was replenished every two days, followed by a day off of treatment for the intermittent dosing schedule. At the end of the incubation period, cell proliferation was measured using the CellTiter 96® nonradioactive cell proliferation assay (MTS) (Promega, France), according to the manufacturer's instructions. HDRAs were conducted in 3 independent experiments.
Nmri foxn1 nu nu mice
Manufactured by Janvier Labs
Sourced in France
The NMRI-Foxn1 nu/nu mice are a strain of laboratory mice with a spontaneous mutation in the Foxn1 gene, resulting in the absence of a functional thymus gland and a lack of mature T cells. These mice are commonly used as models for studying the immune system and evaluating the effects of therapeutic interventions on immune function.
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Lab products found in correlation
Cobimetinib, by Selleck Chemicals (1 mentions)
Dabrafenib gsk2118436, by Selleck Chemicals (1 mentions)
Celltiter 96 non radioactive cell proliferation assay mts, by Promega (1 mentions)
Gelatin, by Merck Group (1 mentions)
Micro fine syringe, by BD (1 mentions)
Growth factor reduced matrigel matrix, by Corning (1 mentions)
8 protocols using nmri foxn1 nu nu mice
1
Patient-Derived Tumor Xenograft Assay
2
Preclinical Pancreatic Cancer Models: Histopathological Characterization and Orthotopic Implantation
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The KPC mouse models have been described previously (33 (link)). The histopathological characterization of KPC tumors into low-grade or W/M differentiated (G1 and G2), and high-grade or poorly differentiated (G3 and G4), was performed at the University Medical Center Göttingen (UMG). For orthotopic mouse models, 1 × 106 human PDAC cells (CAPAN1 and PANC1) were orthotopically implanted into the pancreas of 10-week-old male NMRI-Foxn1nu/nu mice (Janvier Labs), as described previously (30 (link)). Three weeks after tumor cell implantation, a high-resolution ultrasound was performed to monitor tumor size, as described previously (30 (link), 33 (link)). For Figure 7I , this experiment (PANC02-derived C57BL/6 syngeneic orthotopic model; ref. 38 (link)) was performed at UT Southwestern Medical Center. For in vivo studies, CRISPR-mediated dCas9-ROBO3 or LacZ control PANC1 cells were orthotopically implanted into the pancreas of 10-week-old male NMRI-Foxn1nu/nu mice. Mice were sacrificed following body weight loss of more than 20%, at poor overall status, or at experiment endpoint, i.e., 3–5 weeks after detection of a decent sized tumor. Then, tissues were collected for further analysis.
3
In Vivo Mouse Model Study
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All animal experiments were approved by the Animal Welfare Committee and were in accordance with European law. Male NMRI-Foxn1 nu/nu mice (5–8 weeks, Janvier) were housed under standard laboratory conditions (i.e., aseptic condition and 12 h light/dark cycle), given ad libitum access to water and food and acclimated for a week before the start of the experiment.
4
Xenograft Mouse Model for 4SC-202 Efficacy
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For each animal, one million tumor cells were resuspended in 20 μl of a 1:1 mixture of DMEM medium and BD Matrigel Matrix High Concentration (HC), Growth Factor Reduced (GFR) (BD Bioscience) and kept on ice until transplantation. Nine week old virgin NMRI foxn1nu/nu mice (Janvier Labs) were anesthetized by isoflurane inhalation (2–3%, Forene). The cell suspensions were injected under sterile conditions subcutaneously with a 0.3 ml Micro-Fine syringe (BD Bioscience) into the left abdominal flank. After tumors were palpable (size = 100 mm3), mice were randomly divided into two groups (n = 12 per group) as control and treated. Animals were treated with either vehicle (methylcellulose) or 4SC-202 (120 mg/kg in methylcellulose) for 4 days (b.i.d) via oral gavage and then followed for another week until sacrificing. Mouse weight and tumor size were measured daily.
5
Investigating KRAS-driven Tumorigenesis in Nude Mice
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Mice were maintained on a 12-h light/dark cycle with water and food ad libitum throughout the duration of the project. Mouse embryonic fibroblast (MEF) cell lines (5 × 105 cells either KRASWT e.V., KRASWT FSP1-WT, KRASG12D e.V. or KRASG12D shFSP1) were injected in 200 µl PBS into both flanks of 8–10 weeks old male NMRI-Foxn1 nu/nu mice (Janvier). Mice were not randomized. A group size of at least 10 tumors per condition was assumed to achieve significantly different results (p = 0.05) with a power of 80%. For that, cells were harvested from plates using trypsin and washed five times with PBS to remove residual FCS. Mice injected with KRASWT e.V. or KRASWT FSP1-WT were assigned to either vehicle or Liproxstatin-1 treatment groups once tumors reached a minimum size of 2.5 × 2.5 mm. For two consecutive weeks, mice were injected 5 × per week either with vehicle (PBS with 1% DMSO) or Liproxstatin-1 (10 mg/kg). Tumor size was tracked by caliper measurements and volume was calculated as (length × width × width)/2. People performing tumor measurements and calculating tumor volume were blinded to the group allocation. Mice were sacrificed at the end of the treatment and fresh-frozen tumor tissue was used for further analysis.
6
Evaluating Poieskin® Graft Efficacy
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The study was designed to demonstrate the non-inferiority of Poieskin® compared to the reference HSTSG in a relevant animal model (Figure 2 ). Immunosuppressed female NMRI-Foxn1nu/nu mice (aged 7 weeks and weighing 20–23 g) were purchased from Janvier Laboratories (Genest-Saint-Isle, France). They were allowed to acclimatize for 2 weeks before the experiments. Eight mice received a Poieskin® graft, while eight others received HSTSG on their back immediately after a cutaneous excision of 1 × 1.5 cm. The primary endpoint was the area measurement of the graft taken on standardized photographs in a 16-day follow-up. Moreover, the following secondary endpoints were evaluated: area percentage of wound contraction, superficial vascularization assessed by laser Doppler imaging, local inflammation by PET imaging, and histological analysis after euthanasia.
7
Xenograft Prostate Cancer Mouse Model
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The experiments were performed using 7–15 weeks-old male NMRI Foxn1nu/nu mice (Janvier Labs, Le Genest-Saint-Isle, France). Mice were housed in the animal facility of Fraunhofer Institute for Cell Therapy and Immunology (Fraunhofer IZI) according to European (Council Directive 86/609/EEC) and German guidelines (Tierschutzgesetz) for the welfare of experimental animals. All experiments had been approved by the local authorities (Landesdirektion Sachsen TV29/18).
The xenograft prostate cancer model was generated by injection of PC-3 cells as described by Liebscher et al. [22 (link)]. Briefly, 0.5 million PC-3 cells suspended in 100 µL matrigel (Matrigel matrix growth factor reduced, Corning Inc., New York, NY, USA) were transplanted subcutaneously into the right hind leg of mice (Figure 1 B).
The xenograft prostate cancer model was generated by injection of PC-3 cells as described by Liebscher et al. [22 (link)]. Briefly, 0.5 million PC-3 cells suspended in 100 µL matrigel (Matrigel matrix growth factor reduced, Corning Inc., New York, NY, USA) were transplanted subcutaneously into the right hind leg of mice (
8
Athymic Nude Mouse Xenograft Model
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Six-week-old, athymic, female NMRI-Foxn1 nu/nu mice were purchased from Janvier Laboratories (Saint Berthevin, France) and housed under pathogen-free conditions.
Animal procedures were performed in accordance with experimental protocols and guidelines for the care and use of animals and approved by the Strasbourg ethics committee for animal experimentation and the French ministry in charge of research (CREMEAS, Strasbourg, France, Apafis #261).
One week after the mice arrived, SK-HEP-1 cells (10×10 6 ) were injected subcutaneously into the left hindlimb of mice in 50 µL of phosphate-buffered saline. The animal weight and tumor diameter were recorded three times a week during the experiment and followed up to detect any weight loss or change. The small and long axes of the tumor were measured using calipers, and the mean value was recorded as the diameter. Tumor growth occurred over a two-week period, during which a typical diameter of 3-10 mm was observed. Seven days post-cell injection (PCI), 90% of the tumor had reached a suitable diameter (< 6 mm in depth) adapted to the 25 MeV proton beam. The mice were then randomized into several groups of 10 mice to undergo PET exams and immunohistochemical analysis.
Animal procedures were performed in accordance with experimental protocols and guidelines for the care and use of animals and approved by the Strasbourg ethics committee for animal experimentation and the French ministry in charge of research (CREMEAS, Strasbourg, France, Apafis #261).
One week after the mice arrived, SK-HEP-1 cells (10×10 6 ) were injected subcutaneously into the left hindlimb of mice in 50 µL of phosphate-buffered saline. The animal weight and tumor diameter were recorded three times a week during the experiment and followed up to detect any weight loss or change. The small and long axes of the tumor were measured using calipers, and the mean value was recorded as the diameter. Tumor growth occurred over a two-week period, during which a typical diameter of 3-10 mm was observed. Seven days post-cell injection (PCI), 90% of the tumor had reached a suitable diameter (< 6 mm in depth) adapted to the 25 MeV proton beam. The mice were then randomized into several groups of 10 mice to undergo PET exams and immunohistochemical analysis.
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