GCaMP6f-floxed mice (B6;129S-Gt(ROSA)26Sortm95.1(CAG-GCaMP6f)Hze/J) were acquired from Jackson Laboratories (Bar Harbor, MN, USA) and crossed with Kit-Cre mice (c-Kit+/Cre−ERT2), provided by Dr. Dieter Saur (Technical University Munich, Munich, Germany). Kit-Cre-GCaMP6f mice were injected with tamoxifen at 6–8 weeks of age (2 mg for three consecutive days), as previously described (Baker et al., 2021a (link), 2021b (link)). 15 days after tamoxifen injection, Kit-Cre-GCaMP6f mice were anaesthetized by isoflurane inhalation (Baxter, Deerfield, IL, USA) and sacrificed by cervical dislocation. All procedures were approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno. All animals used and the protocols carried out in this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Isoflurane inhalation
Manufactured by Baxter
Sourced in United States, Germany
Isoflurane inhalation is a laboratory equipment product that provides a volatile anesthetic agent. It is used to induce and maintain general anesthesia in laboratory animals.
Automatically generated - may contain errors
Lab products found in correlation
Rnalater, by Qiagen (3 mentions)
C57bl 6, by Jackson ImmunoResearch (3 mentions)
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
C57bl 6n mice, by Charles River Laboratories (2 mentions)
B6.129s gt rosa 26sortm38 cag gcamp3 hze j, by Jackson ImmunoResearch (2 mentions)
B6 129s gt rosa 26sortm95.1 cag gcamp6f hze j, by Jackson ImmunoResearch (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Carprofen, by Zoetis (1 mentions)
Bio gen pro200, by PRO Scientific (1 mentions)
Rnalater, by Merck Group (1 mentions)
12 protocols using isoflurane inhalation
1
Cre-mediated GCaMP6f Expression in Mice
2
Isolation and Preservation of Murine Lymphoid Organs
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First, mice were deeply anaesthetized by isoflurane inhalation (Baxter). Subsequently, mice were transcardially perfused with 60 ml sterile PBS. Single-cell suspension of mesenteric lymph nodes and spleen were generated by mechanically passing tissue through a 40 μm strainer in PBS complemented with 2% fetal calf serum (FCS). Brains were removed and stored in RPMI medium (life technologies) or RNAlater (Qiagen) for additional analysis. Samples stored in RNAlater were kept at 4 °C overnight and then transferred to -20°C. Samples in RPMI medium were stored on ice until further experimental procedures.
3
Murine Model of Bacterial Infection
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Animal experiments were performed with approval of the local State Review Board of Saarland, Germany, and conducted following the national and European guidelines for the ethical and human treatment of animals. Preparation of the bacterial inoculum and infection of the animals were carried out as described [34 (link)], with minor modifications. Briefly, 100 µL bacterial suspensions containing ~107 colony forming units (CFU) were administered intravenously by retro-orbital injection into female, 8- to 10-week-old C57BL/6N mice (Charles River, Sulzfeld, Germany) that were anesthetized by isoflurane inhalation (3.5%; Baxter, Unterschleißheim, Germany). Immediately after infection, mice were treated with a dose of carprofen (5 mg/kg; Zoetis, Berlin, Germany), and at four days post infection, mice were sacrificed, and livers and kidneys were removed. The organs were weight adjusted and homogenized in PBS (Thermo Fisher, Dreieich, Germany), and serial dilutions of the homogenates were plated on blood agar plates to enumerate the CFU rates in the organs.
4
Visualizing Mast Cell Activity in Mice
5
Hippocampal Antioxidant Analysis in Rodents
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At the end of the experiment, the animals were sacrificed by isoflurane inhalation (Baxter Healthcare, Warsaw, Poland). The brains of the animals were excavated and rinsed in saline solution, and then the hippocampi were isolated from both hemispheres. The ventral parts were used for the analyses of total antioxidant status (TAS) and activities of glutathione peroxidase (GPx), glutathione reductase (GSR), and superoxide dismutase (SOD), as well as the level of lipid peroxidation (TBARS) and neurosteroids, while the dorsal parts were used for RNA isolation and gene expression analysis. Hippocampi were frozen in liquid nitrogen and stored at −80 °C for further biochemical analyses. To determine the antioxidant potential, the ventral parts were homogenized in PBS (pH 7.4; Sigma-Aldrich, St. Louis, MO, USA) in a tissue-to-buffer volume ratio of 1:5, using a homogenizer (Bio-Gen PRO 200, PRO Scientific, Oxford, MS, USA). The resulting homogenates were centrifuged for 5 min at 4 °C and 5000× g (Multifuge 3L-R centrifuge, Kendro, Hanau, Germany). After centrifugation, the supernatants were transferred to 200-µL test tubes and frozen at −80 °C for further analyses.
6
Activation of GCaMP3 in ICC Cells
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GCaMP3-floxed mice (B6.129S-Gt(ROSA)26Sortm38(CAG−GCaMP3)Hze/J) and their corresponding wild-type siblings (C57BL/6) were purchased from Jackson Laboratories (Bar Harbor, MN, USA) and subsequently crossed with Kit-Cre mice (c-Kit+/Cre−ERT2) provided by Dr. Dieter Saur (Technical University Munich, Munich, Germany). Kit-Cre-GCaMP3 mice underwent treatment with tamoxifen at 6–8 weeks of age (2 mg for 3 consecutive days), as previously described (Baker et al., 2016 (link)), to induce activation of Cre recombinase in ICC and activate expression of GCaMP3. After tamoxifen (15 days); Kit-Cre-GCaMP3 mice were anesthetized by isoflurane inhalation (Baxter, Deerfield, IL, USA) and killed by cervical dislocation. All animals used for these experiments were handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno [Animal assurance # D16-00311 (A3500-01)].
7
Isoflurane Anesthesia and Euthanasia in C57BL/6N and Ano2-/- Mice
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Eight to twelve weeks old male C57BL/6N mice (Charles River Laboratories, Wilmington, Massachusetts, USA) and Ano2-/- mice [8 (link)] (kindly provided by Dr. Thomas Jentsch, FMP, Berlin, Germany) were used for all the experiments. Mice were kept in the Interfaculty Biomedical Facility (IBF) of the Heidelberg University (Germany) under SPF conditions. Mice were anesthetized by isoflurane inhalation (Baxter, Deerfield, Illinois, USA) and then put to death with an overdose isoflurane. Animal housing and all experimental procedures were carried out in compliance with the guidelines for the welfare of experimental animals as stipulated by the Federal Republic of Germany. Animal experiments were approved by the Regierungspraesidium Karlsruhe, Germany (approval numbers T-04/18, T-05/18).
8
Perfusion and Tissue Harvesting
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Mice were deeply anaesthetized by isoflurane inhalation (Baxter), the CSF was collected and thereafter animals were transcardially perfused with 60 ml PBS. For immunofluorescence samples, perfusion was additionally done with 20 ml of 4% paraformaldehyde (PFA) in PBS. Brain, spleen and spinal cord were removed and stored in sterile ice-cold PBS or RNAlater (Sigma) for further processing. Samples stored in RNAlater were kept at 4 °C overnight and afterwards transferred to − 80 °C.
9
Mesenteric Lymph Node and Spleen Isolation
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First, mice were deeply anesthetized by isoflurane inhalation (Baxter, Halle/Westfallen, Germany). Subsequently, mice were transcardially perfused with sterile ice-cold PBS. Single-cell suspension of mesenteric lymph nodes and spleen were generated by mechanically passing tissue through a 40 μm strainer in PBS complemented with 2% fetal calf serum (FCS). Samples stored in RNAlater (Qiagen, Stockach, Germany) were kept at 4°C overnight and then transferred to -20°C.
10
Xenograft Tumor Induction in Athymic Nude Mice
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