Adult (10–13 week old) male and female K18-hACE2 transgenic mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in groups of 4 in solid–bottom polysulfone individually ventilated cages (Allentown BCU) in rooms maintained on a 12:12-hour light:dark cycle at 22 ± 2°C with 30–70% humidity in the Regional Biocontainment Laboratory (animal biosafety level 3 facility) at UTHSC (Memphis, TN). Mice were acclimated for 1 day before being lightly anesthetized with 2% inhaled isoflurane (Baxter, Deerfield, IL) and implanted subcutaneously with an IPTT300 transponder (Bio Medic Data Systems, Seaford, DE) for identification and temperature monitoring, followed by an additional 3 days of acclimation before inclusion in the experiments. Envigo irradiated rodent diet (catalog no. 7912) and autoclaved water were available ad libitum during the acclimation and study periods; gel food and hydrogel were provided at the time of infection. All experimental procedures were performed under protocol 20–0132 approved by the Animal Care and Use Committee at University of Tennessee Health Science Center (UTHSC) under relevant institutional and American Veterinary Medical Association (AVMA) guidelines and were performed in a biosafety level 3 facility that is accredited by the American Association for Laboratory Animal Science (AALAS).
Inhaled isoflurane
Manufactured by Baxter
Sourced in United States
Inhaled isoflurane is a volatile anesthetic used in laboratory settings. It is a clear, colorless, and odorless liquid that is vaporized and administered through inhalation. Isoflurane is used to induce and maintain general anesthesia in laboratory animals during medical procedures or experiments.
Automatically generated - may contain errors
Lab products found in correlation
Balb c mice, by Jackson ImmunoResearch (2 mentions)
B6.129s4 ccl2tm1rol j mice, by Jackson ImmunoResearch (2 mentions)
Iptt 300 transponder, by Avidity Science (1 mentions)
B6 cg tg k18 ace2 2prlmn j, by Jackson ImmunoResearch (1 mentions)
Vevo 2100 system, by Fujifilm (1 mentions)
Rmv 704, by Fujifilm (1 mentions)
Ob ob b6 cg lepob j, by Jackson ImmunoResearch (1 mentions)
Balb cj mice, by Jackson ImmunoResearch (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
12 protocols using inhaled isoflurane
1
Transgenic Murine Model for SARS-CoV-2
2
Evaluating Virulence of Influenza Virus Variants
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Experiments using 8-week-old female BALB/c mice (Jackson Laboratories, Bar
Harbor, ME) were performed in a Biosafety Level 2 facility in the Animal
Resources Center at St. Jude Children’s Research Hospital (St. Jude;
Memphis, TN) and Centers for Disease Control & Prevention (CDC; Atlanta,
GA). Animals were given general anesthesia that consisted of 2.5% inhaled
isoflurane (Baxter Healthcare Corporation, Deerfield, IL) prior to all
interventions. All studies were approved by the respective institutional animal
care and use committees at St. Jude and CDC. All methods were performed in
accordance with the relevant guidelines and regulations.
To compare the pathogenicity of rgHK68 viruses, mice were infected intranasally
(i.n.) with doses of 102.4 and 105 PFU per mouse
in 50 μl of sterile PBS. Control mice were administered
PBS only. After 1, 3, 5, 7, 8, or 9 days, lungs from 3 to 5 mice
from each group were removed under sterile conditions, washed 3 times with PBS,
homogenized, and suspended in PBS (total volume, 1 ml). The
suspensions for virus titration were centrifuged at
2,000 × g for
10 minutes to clear cellular debris. Virus titers were determined by
TCID50 assays in MDCK cells. Weight changes (calculated for each
mouse as a percentage of its weight on day 0 before virus infection) were
monitored daily for each group (n = 10) for 21 days
after infection.
Harbor, ME) were performed in a Biosafety Level 2 facility in the Animal
Resources Center at St. Jude Children’s Research Hospital (St. Jude;
Memphis, TN) and Centers for Disease Control & Prevention (CDC; Atlanta,
GA). Animals were given general anesthesia that consisted of 2.5% inhaled
isoflurane (Baxter Healthcare Corporation, Deerfield, IL) prior to all
interventions. All studies were approved by the respective institutional animal
care and use committees at St. Jude and CDC. All methods were performed in
accordance with the relevant guidelines and regulations.
To compare the pathogenicity of rgHK68 viruses, mice were infected intranasally
(i.n.) with doses of 102.4 and 105 PFU per mouse
in 50 μl of sterile PBS. Control mice were administered
PBS only. After 1, 3, 5, 7, 8, or 9 days, lungs from 3 to 5 mice
from each group were removed under sterile conditions, washed 3 times with PBS,
homogenized, and suspended in PBS (total volume, 1 ml). The
suspensions for virus titration were centrifuged at
2,000 × g for
10 minutes to clear cellular debris. Virus titers were determined by
TCID50 assays in MDCK cells. Weight changes (calculated for each
mouse as a percentage of its weight on day 0 before virus infection) were
monitored daily for each group (n = 10) for 21 days
after infection.
3
Aortic Diameter Measurement in Mice
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Transthoracic echocardiography (TTE) was performed at age 3 weeks (baseline) and then weekly until age 12 weeks (n = 7‐8). Mice were sedated with 2% inhaled isoflurane (Baxter Healthcare Corporation) delivered via nose cone. The aorta was imaged in the parasternal long‐axis view using a Vevo 770 Imaging Station (VisualSonics Inc) equipped with a Vevo 2100 system and the ultra‐high frequency liner array transducer RMV 704. For statistical analysis, measurement of the aortic diameter (from edge to edge, at the largest portion of the AS aorta) was performed using three technical replicates for each animal by two blinded investigators.
4
Intranasal Infection of BALB/c and C57BL/6 Mice
5
Intranasal Infection of BALB/c and C57BL/6 Mice
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All mouse studies were performed in compliance with animal welfare regulations under an animal protocol approved by the Animal Care and Use Committee of St. Jude Children’s Research Hospital. Seven to eight-week-old female BALB/c mice or C57BL/6 WT and Ccl2−/− (B6.129S4-Ccl2tm1Rol/J mice; Jackson Laboratory) were used for the mouse studies. The animals were lightly anesthetized with 2.5% inhaled isoflurane (Baxter) and afterward infected intranasally with the indicated viruses and plaque-forming units (PFU) in a final volume of 50 μl. Mice were monitored daily for weight loss, disease symptoms, and mortality. In accordance with protection of animal welfare restrictions, mice were sacrificed after weight loss of 30% and considered to have died that specific day.
6
Cardiac Puncture for Murine Tissue Analysis
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Blood was collected by cardiac puncture from mice anesthetized using inhaled isoflurane (Baxter, Deerfield, IL, USA). After clotting on ice, serum was separated by centrifugation, 2 × 8 min at 2100 g, and stored at -20°C before analysis. Following blood collection, mice were killed by cervical dislocation and tissues, including heart, kidney, liver, brain, testis, seminal vesicles, extensor digitorum longus (EDL) muscle, tibialis anterior (TA) muscle, soleus (SOL) muscle, gastrocnemius (GAST) muscle, LA muscle, subcutaneous fat, retroperitoneal fat and gonadal fat, were immediately dissected, weighed and snap-frozen in liquid nitrogen, or fixed in 4% paraformaldehyde. Wet weight of tissues was determined to an accuracy of 0.1 mg, from the mean mass bilaterally. Body and tissue mass were measured in 12 week mAR ΔZF2 male mice and male littermate controls.
7
Determining Viral and Bacterial Infectious Doses
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The viral infectious dose (TCID50) was determined by interpolation using the method of Reed and Muench (94 (link)) using serial dilutions of virus on Madin-Darby canine kidney (MDCK) cells. Bacterial infectious doses (CFU) were determined by serial dilutions on TSA (WT), TSA-ERM (SGD mutants), or TSA-Spec (Tn-seq) plates. Frozen stocks of inocula were diluted in sterile PBS and administered intranasally to groups of 5 (for kinetics) or 10 (for bacteria collection and survival) mice lightly anesthetized with 2.5% inhaled isoflurane (Baxter, Deerfield, IL) in a total volume of 100 μl (50 μl per nostril). Mice were inoculated with either PBS or 75 TCID50 PR8 at day zero and then with 106 CFU of the transposon mutant library, D39, or an SGD mutant (in 100 μl) 7 days later. This dose of bacteria was chosen to ensure that sufficient amounts of bacteria were recovered in Tn-Seq experiments, and the CFU of each inoculum was confirmed as described above. For bacterial sequencing, 500 μl of the inoculum was plated on TSA-Spec plates (100 μl/plate) (t1 in “Bacterial fitness by Tn-Seq”). Animals were weighed at the onset of infection and each subsequent day to monitor illness and mortality. Mice were euthanized if they became moribund or lost 30% of their starting body weight.
8
Mouse Anesthesia and Genotyping
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Male mice aged 7 to 10 weeks (weight 20-25 g) were bred and maintained in the Montreal General Hospital animal facility. C57BL/6 mice were obtained from Harlan Labs (Indianapolis, IN). TLR4-negative/negative, TLR2-negative/negative, and MyD88-negative/negative mice (against a C57BL/6 background) were obtained from Dr. Michel Chignard (Pasteur Institute, Paris, France). Subsequently, their genotypes were verified by ear punch DNA polymerase chain reaction analysis. Mice were anesthetized as previously described 13 or through inhaled isoflurane (Baxter, Mississauga, Canada) or with a mixture of 200 mg/kg of ketamine and 10 mg/kg of xylazine by intraperitoneal injection. All experiments were approved by the McGill University Animal Care Committee and were conducted in accordance with institutional guidelines.
9
Coinfection and Metabolic Disease Impacts
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Coinfection experiments were performed as described previously (14 (link)). Briefly, 8-week-old C57BL/6 (lean) and B6.Cg-Lepob/J (ob/ob) mice (Jackson Laboratory, Bar Harbor, ME) along with diet-induced lean and obese mice were anesthetized with 2.5% inhaled isoflurane (Baxter, Deerfield, IL) and intranasally inoculated with sublethal doses of influenza virus in a total volume of 30 µl. Three, 7, or 10 days after influenza virus infection, mice were anesthetized with 2.5% inhaled isoflurane and intranasally inoculated with 100 CFU of S. pneumoniae, and then monitored daily for clinical signs of infection and weighed every 24 hours postinfection (59 (link)). Moribund mice that had lost more than 30% body weight were humanely euthanized. At days 7, 8, 9, and 10 postinfection, mice were euthanized by CO2 asphyxiation, and tissues (lung, bronchoalveolar lavage fluid, nasal wash, blood) were harvested and processed immediately or stored at −80°C for future analysis.
10
Bacterial Pneumonia Mouse Model
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