At gestational day 119, pregnant gilts underwent Cesarean sections. We chose gestational day 119 instead of 114 (normal gestation) because piglets derived from somatic cell nuclear transfer typically requiring longer gestational period. Initial anesthesia was induced with either a lumbar epidural of propofol (0.83–1.66 mg/kg) (Zoetis) or intravenous injection of Ketamine (1–2 mg/kg) (Akorn) and Xylazine (1–2 mg/kg) (Akorn), and anesthesia was maintained on oxygen and isoflurane (Phoenix). An abdominal incision was made to expose and remove the uterus. After removal, the uterus was immediately rinsed in chlorohexidine and then surgically opened to remove the piglets. All piglets had their cords clamped before being immediately placed into sterile polystyrene boxes and delivered into biocontainment facilities (14 (link)). All piglets were fed ~250 mL of pasteurized porcine colostrum within the first 24 h of life. A total of eight Art−/−IL2RG−/Y SCID pigs were created and assessed within this study (animal IDs: 6401, 6402, 6403, 6701, 6702, 6901, 6902, and 6903). Piglets derived from the gilt that underwent laparotomy procedures for human stem cell injection (see below) did not receive colostrum. After birth, DNA was isolated from ear notch tissues and subjected to genotyping for ART and IL2RG status using primers and protocols described in Supplemental Table 1 .
Ketamine
Manufactured by Akorn
Sourced in United States, China
Ketamine is a general anesthetic medication used in medical procedures. It is a dissociative anesthetic that induces a trance-like state while providing pain relief, sedation, and amnesia. Ketamine is primarily utilized in veterinary medicine and hospital settings for its anesthetic properties.
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Lab products found in correlation
Xylazine, by Akorn (11 mentions)
Cefazolin, by Qilu Pharmaceutical (3 mentions)
Propofol, by Fresenius (2 mentions)
Baytril, by Bayer (2 mentions)
Buprenex, by Reckitt Benckiser (2 mentions)
Tropicamide, by Akorn (2 mentions)
Nicotine tartrate salt, by MP Biomedicals (2 mentions)
Propofol, by Zoetis (1 mentions)
Isoflurane, by Phoenix Pharmaceuticals (1 mentions)
Live dead bacterial viability assay, by Thermo Fisher Scientific (1 mentions)
Euthasol, by Virbac (1 mentions)
Midazolam, by Akorn (1 mentions)
Micron 3 retinal imaging microscope, by Phoenix Pharmaceuticals (1 mentions)
Systane, by Alcon (1 mentions)
Tropicamide, by Bausch & Lomb (1 mentions)
Genteal, by Alcon (1 mentions)
Proparacaine, by Akorn (1 mentions)
Spectralis hra oct system, by Heidelberg Engineering (1 mentions)
Uniprim, by Inotiv (1 mentions)
Espion erg equipment, by Diagnosys (1 mentions)
15 protocols using ketamine
1
Generating SCID Pigs from Somatic Cells
2
In vivo Bacterial Killing Assay in Pigs
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Pigs were sedated intramuscularly with ketamine (20 mg/kg) and xylazine (2 mg/kg) (both Akorn Animal Health, Lake Forest, IL, USA) and maintained with intravenous propofol (Fresenius Kabi USA, Lake Zurich, IL, USA; 1 mg/kg) for the pH and bacterial killing assays. During anesthesia, oxygen levels, temperature, pulse, and respiratory rate were monitored. Pigs were euthanized using Euthasol (Virbac, Forth Worth, TX, USA; 90 mg/kg). The bacterial killing assay was developed by Pezzulo et al. (1 (link)). Briefly, S. aureus (strain SA43) was immobilized on gold electron microscopy grids through a streptavidin and biotin interaction. Bacterial-coated grids were placed on the tracheal surface of a sedated pig for 1 min and queried for cell death by a propidium iodide stain (Live/Dead Bacterial Viability Assay, Invitrogen, Carlsbad, CA, USA). Grids were imaged by confocal microscopy and analyzed using ImageJ (ImageJ, Schneider, CA, USA).
3
Compression Spinal Cord Injury Model
4
Compression Spinal Cord Injury Model
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The impact of HFHS consumption on recovery after SCI was examined using an experimental model of compression SCI (Plemel et al., 2008 (link)). Prior to spinal cord compression injury, mice were deeply anesthetized with xylazine (10 mg/kg; Akorn, Inc. Lake Forest, IL, USA) and ketamine (100 mg/kg; Fort Dodge, IA, USA). A laminectomy was performed leaving the dura intact and Dumont #2 forceps with a 0.25 mm spacer was applied to laterally compress the spinal cord at T8–9 for 14 s. Immediately after surgery, 1 ml of saline was administered intraperitoneally to replace lost blood volume. Baytril (10 mg/kg; Bayer Healthcare, LLC; Animal Health Division; Shawnee Mission, Kanas, USA) was delivered once immediately post-surgically. Buprenex (0.05 mg/kg; Reckitt Benckiser Healthcare, England) were delivered every 12 h for 3 d post-operatively to minimize infection risk and pain, respectively. Bladders of injured mice were manually vacated twice daily until recovery of spontaneous bladder release. All experiments were approved by the Mayo Clinic Institutional Animal Care and Use Committee and completed in accordance with NIH Guidelines.
5
Tissue Collection for Primate Research
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Blood serum, fixed liver tissue, and frozen liver tissue were obtained from shared control animals from various ongoing studies, as described below. Animals (Papio anubis and Papio cynocephalus x Papio anubis hybrids) were obtained from the Texas Biomedical Research Institute (TBRI) in San Antonio, Texas and the Oklahoma Primate Center (Fort Reno, OK). Experiments were conducted at the TBRI and at the University of Texas Health Science Center at San Antonio (UTHSCSA, San Antonio, TX). The Institutional Animal Care and Use Committees at the TBRI and UTHSCSA approved all studies. Animal experiments were conducted in accordance with accepted standards of humane animal care, and all efforts were made to minimize suffering. There were no early deaths of preterm or term animals. Prior to euthanasia, preterm animals were anesthetized with Ketamine (5 mg/kg, Putney, Portland, USA) and Midazolam (0.1mg/kg, Akorn, Lake Forest, USA) IV and term animals were anesthetized with Ketamine (10mg/kg; IM) and isoflurane gas (1–2%) and titrated to effect. All animals were euthanized via exsanguination followed by pentobarbital. Necropsies were performed immediately following euthanasia.
6
Mouse Retinal Imaging Protocol
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Mice were anesthetized with a solution of 50 mg/mL ketamine (Akorn, Lake Forest, IL) and 5 mg/ml xylazine (Lloyd Laboratories, Shenandoah, IA) using 2 μl/g mouse. Pupil dilation was done with 2.5 μl of (0.5%) tropicamide (Bausch and Lomb, Tampa FL) and (0.25%) proparacaine (Akorn) solution applied topically to the cornea. Corneal hydration was maintained by liberal application of Systane (Alcon, Fort Worth, TX) or GenTeal (Alcon). Retinal images were obtained using a Micron III retinal imaging microscope (Phoenix Research Laboratories, Pleasanton, CA). White light (brightfield) and fluorescence image were obtained. For GFP, a 469/35 nm band pass excitation filter and a 525/50 nm band pass emission filter were used. For Tomato Red and RFP the excitation filter was 562/40 nm and the emission filter was 624/40 nm.
7
Retinal Microarchitecture Imaging in Rodents
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OCT images of the retinal microarchitecture were obtained using the Spectralis HRA + OCT system (Heidelberg Engineering Inc., Heidelberg, Germany). Animals were anesthetized with xylazine (10 mg/kg, Akorn) and ketamine (100 mg/kg, Akorn, Lake Forest, IL). Pupils were dilated using 0.5% tropicamide (Akorn), and contact lens was used to prevent corneal dehydration. While anesthetized, animals were kept on a heating pad (37 °C) to prevent hypothermia. OCT volume scans (30° × 25° degrees with 61 individual b-scans, 120 µm distance between B-scans) were obtained, centered on the optic nerve head of each eye (left eye, HI injured; right eye, control) using the instrument’s automatic real-time tracking mode (ART) averaging ten frames per b-scan.
8
Nerve Graft Repair in Lewis Rats
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The University of Wisconsin-Madison Animal Care and Use Committee (ACUC, protocol# M5958) approved all procedures on January 3, 2018, which followed the NIH Guide for animal care. Seventy-two inbred male Lewis rats weighing ~250 g were used (Envigo, Huntingdon, UK). The inbred Lewis rat strain enabled the use of a nerve graft from a donor rat without the need of immunosuppression (Hellenbrand et al., 2016). For all surgical procedures, an intraperitoneal injection of 90 mg/kg ketamine (Akorn, Inc., Lake Forest, IL, USA) and 9 mg/kg xylazine (Bimeda, Oakbrook Terrace, IL, USA)was given for the anesthetic agent. Following surgical procedures, the rats received subcutaneous injections of Carprofen (Rimadyl) 5 mg/kg to control pain. They also received an antibiotic enriched diet of Uniprim (Envigo, Huntingdon, UK; Cat# TD.06596), for 7 days following surgery. All rats were given food and water ad libitum, housed at room temperature with two rats per cage and standard light/dark cycle.
9
Generation and Characterization of CF Pigs
10
Murine Retinal Electrophysiology Using ERG
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