Male C57BL/6 (8–9 weeks old) mice were purchased from Vital River (Beijing, China). The human breast cell lines (BCap37, Hs 578T, MDA-MB-231, MDA-MB-468, BT-474, SK-BR-3, MCF-7, Hs 578Bst and HBL-100) were purchased from the cell bank of the Chinese Scientific Academy. BCap37, BT-474 and MCF-7 cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, 31800105, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Biological Industries, 04-0101-1, Cromwell, CT, USA). MDA-MB-231 and MDA-MB-468 cells were cultured in Leibovitz's L-15 medium (Gibco, 11415114) with 10% FBS. Hs 578T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, 12430047) supplemented with 0.01 mg/ml bovine insulin (Solarbio, I8040, Beijing, China) and 10% FBS. SK-BR-3 cells were cultured in McCoy's 5A medium (modified, Gibco, 16600082) with 10% FBS. Hs 578Bst cells were cultured in DMEM with 50 ng/ml epidermal growth factor (Gibco, PHG0311) and 15% FBS. HBL-100 cells were cultured in DMEM with 10% FBS. The cell culture medium was changed every 2–3 days, and the cells were passaged with 0.25% trypsin-EDTA (Gibco, 25200056) and grown to 90% confluence. The cultures were kept at 37 °C with 5% CO2 in a water-jacketed incubator (Thermo Scientific, Waltham, MA, USA).
Male c57bl 6
Manufactured by Charles River Laboratories
Sourced in China, United Kingdom, Germany
Male C57BL/6 is a commonly used mouse strain that serves as a standard laboratory model. This inbred strain is known for its well-characterized genetic background and is widely utilized in various research applications.
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Lab products found in correlation
Mda mb 231, by Thermo Fisher Scientific (1 mentions)
Sk br 3, by Thermo Fisher Scientific (1 mentions)
Bt474, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Mcf 7, by Thermo Fisher Scientific (1 mentions)
Mda mb 468, by Thermo Fisher Scientific (1 mentions)
Hs578t, by Thermo Fisher Scientific (1 mentions)
Bcap37, by Thermo Fisher Scientific (1 mentions)
Water jacketed incubator, by Thermo Fisher Scientific (1 mentions)
Bovine insulin, by Solarbio (1 mentions)
Male c3h mice, by Charles River Laboratories (1 mentions)
Male balb c, by Charles River Laboratories (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Fluoxetine, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Cox 2, by Santa Cruz Biotechnology (1 mentions)
12 protocols using male c57bl 6
1
Culturing and Maintaining Human Breast Cell Lines
2
Coxsackievirus B3 Infection Model in Mice
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This study was approved by the Institutional Animal Research Committee of Tongji Medical College. All animal experimental protocols complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Male A/J (~6-week-old) mice were obtained from GemPharmatech (Nanjing, China). Male C57BL/6 (~6-week-old) mice, male BALB/c (~6-week-old) mice, and male C3H mice were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). All four mice strains were randomly assigned to two groups: control (n = 10) and CVB3-treated (n = 15) groups. A/J mice and BALB/c mice were intraperitoneally injected with 104 PFU CVB3, while C57BL/6 and C3H mice were intraperitoneally injected with 105 PFU CVB3, as described previously (Huber et al., 2001 (link); Althof et al., 2018 (link)). Phosphate-buffered saline (PBS) was used as the control. All animals were anesthetized with intraperitoneal injections of a mixture of xylazine (5 mg/kg) and ketamine (80 mg/kg). Echocardiography was performed on day 7 after virus injection, and the mice were sacrificed. Subsequently, the organs were collected and frozen in liquid nitrogen, followed by storage at −80°C.
3
Murine Osteoarthritis Model using MCP-1
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Male C57Bl/6 (Vital River Laboratory) mice aged 10 weeks were used in all experiments. The right knee joints of the mice were injected once a week, for 3 weeks, intra-articularly through the patellar ligament, with a 6-μl solution of MCP-1 (CB500038, California Bioscience, Coachella, CA, USA) at a concentration of 25 ng/ml. The dissolving agent was 0.1 % BSA, according to the manufacturer. At the same time, the left control knee joint was injected with 6 μl of 0.1 % BSA. Two mice, injected with MCP-1, were sacrificed by cervical dislocation after an inhalation of ibuprofen at two time-points 21 and 42 days, after intra-articular injection. Histological assessments of knee joints in the mouse were adopted from the OARSI histopathology initiative [13 (link)].
4
Avicularin Modulates Inflammatory and Apoptotic Pathways
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Male C57BL/6 (age 8–10 weeks) mice weighting 18–22 g were purchased from the Vital River Company (Beijing, China). Animals were housed under a standard environment (temperature, 22±2°C, 55±5% humidity and 12-h light/dark cycle) and allowed food and water ad libitum. All experimental procedures were conducted in accordance with the Institutional Animal Care and Use Committee at Jiaxing University and carried out in accordance with the National Guidelines for Animal Care and Use Committees. The present study was approved by the Animal Ethics Committee of Jiaxing University.
The following reagents were used. Avicularin (purity >99%) was purchased from Aladdin Chemistry Company (Shanghai, China). Fluoxetine was obtained from Sigma-Aldrich. The primary antibodies used were: phosphorylated-MEK1/2 (p-MEK1/2) (Cell Signaling Technology, 1: 1000), p-ERK1/2 (Cell Signaling Technology, 1: 1000), phosphorylation of NF-κB p65 (p-p65, Proteintech, 1: 500, USA), iNOS (Cell Signaling Technology, 1: 1000), COX-2 (Santa Cruz Biotechnology, 1: 1000), Caspase-3 (Cell Signaling Technology, 1: 1000), Bax (Cell Signaling Technology, 1: 1000), and β-actin (Sigma, 1: 1000).
The following reagents were used. Avicularin (purity >99%) was purchased from Aladdin Chemistry Company (Shanghai, China). Fluoxetine was obtained from Sigma-Aldrich. The primary antibodies used were: phosphorylated-MEK1/2 (p-MEK1/2) (Cell Signaling Technology, 1: 1000), p-ERK1/2 (Cell Signaling Technology, 1: 1000), phosphorylation of NF-κB p65 (p-p65, Proteintech, 1: 500, USA), iNOS (Cell Signaling Technology, 1: 1000), COX-2 (Santa Cruz Biotechnology, 1: 1000), Caspase-3 (Cell Signaling Technology, 1: 1000), Bax (Cell Signaling Technology, 1: 1000), and β-actin (Sigma, 1: 1000).
5
Surgical Procedure in Aged Mice
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Male C57BL/6 aged 14-16 months old was purchased from Beijing Vital River Laboratory Animal Technology (China). The mice were housed under specific pathogen-free conditions (ambient temperature, 22.0 ± 1.0°C, and humidity, 40%) during breeding and the experiments. Food and water were available ad libitum. All experimental procedures involving animals were approved by the Animal Care and Use Committee of Ningbo University in following guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health.
The mice were randomly assigned to one of four groups: control (con), surgery (sur), surgery + VD3 (sur + VD3), and surgery + vehicle (sur + veh). Half of the animals in each group underwent behavioral testing while the other half were sacrificed 24 hours following the surgery for molecular detection to minimize the potential confounding effects of behavioral tests on inflammatory markers (Figure 1 ).
The mice were randomly assigned to one of four groups: control (con), surgery (sur), surgery + VD3 (sur + VD3), and surgery + vehicle (sur + veh). Half of the animals in each group underwent behavioral testing while the other half were sacrificed 24 hours following the surgery for molecular detection to minimize the potential confounding effects of behavioral tests on inflammatory markers (
6
Mouse Handling and Care Protocol
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Male C57BL/6 (6–8 weeks, 20–22 g) mice were purchased from Charles river (Beijing, China). All animal care and experimental procedures complied with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Nankai University (Permit No. SYXK 2014–0003). All experimental protocols were approved by the Animal Experiment Committee of Tianjin International Joint Academy of Biomedicine (approval no. SYXK (JIN) 2017–0003).
7
Murine Model Experiments Protocols
8
Neutrophil-Deficient Bone Marrow Chimera
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Male C57BL/6 (Charles River, Germany) and bone marrow chimera mice (see below) weighing 25.4 ± 3.8 g were maintained under standardized conditions (12:12 h light-dark cycle, 40–70% relative humidity, and constant ambient temperature of 22 ± 2 °C), with free access to standard rodent chow (Akronom Kft., Budapest, Hungary) and tap water. The experiments were approved by the Animal Ethics Committee of Semmelweis University and the Pest County Government Office (PE/EA/2202-5/2017). All procedures were performed in accordance with the Hungarian Law on the protection and welfare of animals and the EU Directive 2010/63/EU for animal experiments.
For testing the role of neutrophil granulocytes, bone marrow chimeras with a neutrophil-deficient Mcl1flox/floxLysMCre/Cre hematopoietic system were used [47 (link),48 (link)]. Briefly, C57BL/6 recipients were irradiated with 11.5 Gy gamma rays using a 137Cs source and injected intravenously with unfractionated bone marrow cells from wild-type (WT) or Mcl1flox/floxLysMCre/Cre (referred to as Mcl-1ΔMyelo) mice on C57BL/6 background. The number of circulating neutrophils was counted by flow cytometry four weeks later. Neutrophils were defined as Ly6G-positive cells within a typical forward and side-scatter gate.
For testing the role of neutrophil granulocytes, bone marrow chimeras with a neutrophil-deficient Mcl1flox/floxLysMCre/Cre hematopoietic system were used [47 (link),48 (link)]. Briefly, C57BL/6 recipients were irradiated with 11.5 Gy gamma rays using a 137Cs source and injected intravenously with unfractionated bone marrow cells from wild-type (WT) or Mcl1flox/floxLysMCre/Cre (referred to as Mcl-1ΔMyelo) mice on C57BL/6 background. The number of circulating neutrophils was counted by flow cytometry four weeks later. Neutrophils were defined as Ly6G-positive cells within a typical forward and side-scatter gate.
9
Standardized Housing for C57BL/6 Mice
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Male C57BL/6 (Charles River Laboratories, Sulzfeld, Germany) mice were maintained under standardized (light on 08:00–20:00 h; 40–70% relative humidity, 22 ± 1 °C), specified pathogen-free (SPF) conditions, with free access to standard rodent chow (Altromin standard diet, Altromin International, Lage, Germany) and tap water. All procedures were performed in accordance with guidelines set by the National Institutes of Health (USA), the Hungarian law on animal care and protection. All protocols were approved by the Committee on Animal Welfare of Semmelweis University and the Pest County Government Office (registration numbers: XIV-I-001/2103–4/2012 and 22.1/321/3/2011).
10
Age and Genotype Effects on Mice
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Male C57BL6 [Charles River, Margate, Kent, UK] wild type and homozygote β 2-AR knockout mice in mixed C57BL/6J/FVB/N background, bred in the laboratory in Würzburg, were used [14] . The age of the mice ranged from 9 to 15 months and their body weight ranged from 20-30 g. All mice were group-housed in cages with free access to food and water at least 4 days before the experiments were conducted.
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