The animals were maintained until the PND89, when euthanasia was performed. Animals were deeply anesthetized with a lethal dose of ketamine cocktail 80 mg/Kg ketamine (100 mg/mL—Syntec), 15 mg/Kg xylazine (20 mg/mL—Syntec) and 1 mg/Kg acepromazine (2 mg/mL—Vetnil) and intracardially perfused (infusion pump Cole-Parmer/Masterflex; model 7518-00), through the ascending aorta with 0.9% saline solution and 4% cold paraformaldehyde. The brain tissues were fixed in 4% paraformaldehyde for 24 h followed by immersion in a 30% sucrose in 0.1 M phosphate buffer at 4°C for 72 h. Brain tissues were frozen in O.C.T. compound (A.O. Company, Milwaukee, WI, USA), and the organs were cut into 30 μm coronal sections on a cryostat (HYRAX C25 cryostat, Zeiss). The sections were stored in a cryoprotectant solution (30% sucrose, 30% ethylene glycol, 0.1 M phosphate buffer) at −80°C until processing for superoxide detection. We decided to select the dentate gyrus of the hippocampus because this is the most sensitive region to damage and neuronal death caused by oxidative stress.
Ketamine
Manufactured by Syntec
Sourced in Brazil
Ketamine is a general anesthetic used in various medical and veterinary applications. It is primarily used to induce anesthesia and provide pain relief. The core function of Ketamine is to act as a dissociative anesthetic, which means it can temporarily separate the perception of pain from the central nervous system.
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Lab products found in correlation
Xylazine, by Syntec (38 mentions)
Xylazine, by Ceva (3 mentions)
Phycoerythrin pe conjugated anti ly6g, by Thermo Fisher Scientific (2 mentions)
Sytox green, by Thermo Fisher Scientific (2 mentions)
Carrageenan, by Merck Group (2 mentions)
Xylasine, by Syntec (2 mentions)
Isoniazid, by Merck Group (2 mentions)
Uric acid, by Merck Group (1 mentions)
Detomidine, by Syntec (1 mentions)
Xylazine, by Bayer (1 mentions)
Ketamine, by Bayer (1 mentions)
Xilazine, by Syntec (1 mentions)
Collagenase 7, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Ultra 5 block reagent, by Thermo Fisher Scientific (1 mentions)
Evans blue dye, by Merck Group (1 mentions)
Flunixin meglumine, by Merck & Co (1 mentions)
Lidocaine, by Dentsply (1 mentions)
Mp100, by Biopac (1 mentions)
Ketamine xylazine, by Syntec (1 mentions)
57 protocols using ketamine
1
Euthanasia and Brain Tissue Fixation
2
Murine Model of Gouty Arthritis
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Crystals of MSU was prepared from uric acid (Sigma-Aldrich - St. Louis MO, United States) as previously described (Amaral et al., 2012 (link)). Mice under anesthesia (80:15 mg/kg ketamine:xylazine; i.p., Syntec, São Paulo, Brazil) were injected into the tibiofemoral knee joint with 100 μg of MSU crystals. The selective PI3K-γ inhibitor (AS605240 – Echelor), the selective PI3K-δ inhibitor [GSK045, (Gupta et al., 2016 (link); Khan et al., 2017 (link)), kindly donated by GlaxoSmithKline - GSK], the PI3Kγ/δ inhibitor [CL27c, a pan-PI3K Inhibitor – (Pirali et al., 2017 (link))] or vehicle were given locally (intraarticular injection) at 12 h after the injection of MSU crystals (see Figure 1 ). Inflammatory parameters were evaluated at different time points after treatment, as indicated in each figure legend. Mice were euthanized and the knee cavity washed with PBS/BSA 3% (2 × 5 μL) to collect the cells. The total number of leukocytes were determined using the newbauer chamber after staining with Turk’s solution. The differential counts were performed using standard morphologic criteria on a slide stained with May-Grunwald-Giemsa. Periarticular tissues were collected from the joints for evaluation of cytokines and myeloreproxidase (MPO) activity.
3
Anesthetic Induction Procedure in C57Bl/6 Mice
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6–8-week-old male C57Bl/6 mice were obtained from “Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia” (CEDEME/UNIFESP, Brazil) animal facility and maintained with a 12:12 h light:dark cycle and water ad libitum. Experiments were performed after anesthetic induction with ketamine (100 mg/kg; Syntec, Brazil) and xylazine (10 mg/kg; Syntec). Mice were euthanized by anesthetic overdose. All procedures were in accordance with the guidelines of the US National Research Council for care and use of laboratory animals [16 ].
4
Bilateral Ovariectomy and Post-Surgical Care
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The mice underwent surgical bilateral ovariectomy through a dorsal incision under anesthesia with 100 mg/kg of ketamine and 10 mg/kg of xylazine (Syntec®, Brazil). Additional anesthetic doses were given throughout the procedure as needed to maintain a constant level of anesthesia, determined by pupil constriction and absence of reflexive withdrawal of the hind limbs, indicative of adequate anesthesia. After removal of the ovaries, the dorsal wall was sutured and the cutaneous incisions closed with 10-mm calipers. As prophylaxis, animals received the anti-inflammatory flunixin meglumine (EquiMed Staff®, USA) at a dose of 2 mg/kg prior to surgery (im). The experimental procedures started 15 days after surgery.
5
Standardized Wound Creation in Rodents
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For wound creation, the animals were anesthetized with Ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally. Ketamine and xylazine were obtained from Syntec®.The dorsal region of each animal was trichotomized and four wounds were produced in this region with a 5 mm punch. The wounds were made equidistant with about 1 cm of distance between them. All animals were housed in individual cages after surgery avoiding any external intervention that could compromise the results.
6
Intestinal Mucositis Intervention Protocols
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Mice were split into six experimental groups (n = six animals per group): negative control (NC), inflamed (MUC), inflamed (5-FU), inflamed and treated with viable CIDCA 133 (LDv), heat-killed inactivated CIDCA 133 (LDi), and CIDCA 133 cell-free supernatant (LDs). Mice received doses of 300 μL containing 109 CFU/mL of CIDCA 133 (LDv and LDi groups) or 300 μL of CIDCA 133 supernatants (LDs group) for 13 days by intragastric gavage. Control groups received 300 μL of PBS 0.1M solution (NC and MUC groups) or MRS broth (5-FU group) by the same route. On the 10th day, mice (MUC, 5-FU, LDv, LDi, and LDs groups) were inflamed intraperitoneally with a single injection of 5-Fluorouracil (Fauldfluor®) (300 mg/kg) (Libbs, São Paulo, Brazil) [29 (link)]. Sterile saline solution injection (NaCl 0.9%) (Vetec, Rio de Janeiro, Brazil) (Figure 1 ) was administered to the negative control group. At the end of the experimental procedure, 72 h after mucositis induction, mice were euthanized by an anesthetic overdose (ketamine 300 mg/kg and xylazine 30 mg/kg solution) (Syntec, Tamboré, Brazil), and afterward, ileum sections were collected for analysis.
7
Probiotics Modulate Inflammatory Bowel Disease
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Mice were randomly split into three non-inflamed groups: Negative control (NC), L. lactis pExu: empty (pExu), L. lactis pExu:p62 (P62), and three inflamed groups: Inflamed DSS 2% (DSS), L. lactis pExu: empty (DSS-pExu) and L. lactis pExu:p62 (DSS-P62). During the experimental period, NC and DSS groups received 300 μL of phosphate-buffered saline (PBS, 0.1 M) solution daily by gavage for five consecutive days. In the same period, the other groups received daily treatments (300 μL) with L. lactis NCDO2118 (pExu: empty or pExu:p62; 109 CFU/animal/day). After 10 days of intermission, DSS, DSS-pExu, and DSS-P62 groups were inflamed by the addition of DSS 2% in the drinking water (MP Biomedicals, Lot: S6132, Cat: 160110) provided ad libitum for 7 days. NC, pExu, and P62 groups received sterile water in the same period. Liquid and chow intake were measured during all experiment periods. On the 22nd day, mice were anesthetized by a single intraperitoneal injection (16 mg/kg of xylazine and 80 mg/kg of ketamine, Syntec, Tamboré, Brazil; i.p) for blood collection via an axillary vein. Afterward, the animals were euthanized by cervical dislocation, and liver, spleen, and colon tissues were harvested and stored immediately at −80°C (Figure 1 ).
8
Breeding and Rearing Rhodnius prolixus
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Rhodnius prolixus were obtained from a colony maintained by the Vector Behavior and Pathogen Interaction Group at René Rachou Institute (FIOCRUZ, Minas, Brazil), which was established from insects collected in Honduras around 1990. The colony was maintained under controlled temperature (26 ± 1°C), relative humidity (65 ± 10%), and natural illumination cycle. Insects were consistently fed on diverse sources of blood that included mice, chicken, and a membrane feeder offering citrated rabbit blood at 37°C. Mice and chickens were anesthetized with intraperitoneal injections of ketamine (150 mg/kg; Cristália, Brazil) plus xylazine (10 mg/kg; Bayer, Brazil), and ketamine (20 mg/kg; Cristália, Brazil) plus detomidine (0.3 mg/kg; Syntec, Brazil), respectively. Rhodnius prolixus 4th instar nymphs starved for 30 days were used in all experiments.
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Rhodnius prolixus were obtained from a colony maintained by the Vector Behavior and Pathogen Interaction Group at René Rachou Institute (FIOCRUZ, Minas, Brazil), which was established from insects collected in Honduras around 1990. The colony was maintained under controlled temperature (26 ± 1°C), relative humidity (65 ± 10%), and natural illumination cycle. Insects were consistently fed on diverse sources of blood that included mice, chicken, and a membrane feeder offering citrated rabbit blood at 37°C. Mice and chickens were anesthetized with intraperitoneal injections of ketamine (150 mg/kg; Cristália, Brazil) plus xylazine (10 mg/kg; Bayer, Brazil), and ketamine (20 mg/kg; Cristália, Brazil) plus detomidine (0.3 mg/kg; Syntec, Brazil), respectively. Rhodnius prolixus 4th instar nymphs starved for 30 days were used in all experiments.
9
Evaluating Toxicity of V. polysphaera Extract in Mice
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The mice were placed in 4 groups (n = 5): treated with PBS (Control), 50, 250 or 500 mg/kg of V. polysphaera extract in a final volume of 0.1 mL. Treatment was administered daily by gavage for 14 days. Clinical signs of toxicity, such as piloerection, diarrhea, salivation, convulsions or changes in mobility, respiration rate or muscle tone, as well as mortality were observed during the treatment. On the 15th day, the animals were euthanized with the 250 μL intraperitoneal injection of a 1:1 mixture of ketamine (100mg/mL; Syntec, BRA) and xylazine (20mg/mL; Syntec, BRA). Whole blood was collected by cardiac puncture to obtain serum for the biochemical analysis of creatinine, total bilirubin, uric acid, albumin, urea, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total protein. Macroscopic evaluation was carried out to check for abnormal findings in the stomach and gut mucosa.
10
Toxoplasma gondii Reactivation Study in Mice
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BALB/c and C57BL/6 mice (5 mice per group) were infected i.p. with 10 cysts of ME49 T. gondii and sacrificed on days 7, 32 and 56 post-infection (p.i.). In order to evaluate T. gondii reactivation, another mouse group (5 mice per group) was treated with 10 mg/mL dexamethasone phosphate in drinking water from 32 to 56 days p.i. [19] (link). During treatment, mice were observed for weigh change and morbidity scores [20] (link). Mice were anesthetized (ketamine and xilazine; Syntec, Brazil), the blood was collected and they were sacrificed for tissue sample collection. Brain, lung, liver and spleen tissue samples were processed in two ways: (1) fixed in 10% buffered formalin and embedded in paraffin for histological procedures or (2) immediately frozen at -80°C for further PCR mRNA quantification.
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