difficile infection? All animal experiments were INCB018424 mouse conducted with the approval of the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan (Protocol Number: 10212). The University’s animal-care policies follow the Public Health Service policy on Humane Care and Use of Laboratory Animals. The mice were housed in an AAALAC-accredited facility. None of the conducted experiments involved
the deliberate induction of discomfort or injury. The physical condition and behaviour of the mice were assessed on a daily basis. The mice were killed by CO2 asphyxiation in compliance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. C57BL/6 mice obtained from Jackson Laboratories (Bar Harbor, ME) were used to establish a breeding colony at the University of Michigan Medical School. They were housed under specific pathogen-free conditions and consumed clean food and water ad libitum. Male mice at 5–8 weeks of age were used for the current set of experiments. The mouse model of C. difficile infection described by Chen et al.[33] was used for this study. Male mice, 5–8 weeks old, were either left untreated Venetoclax order or received an antibiotic mixture of colistin (850 U/ml), gentamicin (0.035 mg/ml), kanamycin (0.4 mg/ml), metronidazole (0.215 mg/ml) and vancomycin (0.045 mg/ml) in sterile drinking water for 3 days. The mice receiving
the antibiotic cocktail were then switched to regular drinking water for 2 days. Afterwards, each of the treated mice was given a single intraperitoneal dose of clindamycin (10 mg/kg) a day before infection with C. difficile. The C. difficile strain used in this study was the reference strain VPI 10463 (ATCC 43255), which was grown and prepared for inoculation as previously described.[35] Each mouse received selleck kinase inhibitor 105 colony-forming units (CFU) of the bacterium in its vegetative state by oral gavage. All the animals were monitored for signs of disease including diarrhoea, hunched posture and weight loss. All untreated and C. difficile-infected mice were killed 42 h after the infection (Fig. 1). Intestinal leucocyte enrichment was performed as previously described,[14, 37] with certain modifications. The caecum and colon
of each mouse were excised, opened longitudinally and washed in PBS to remove the faecal content. Afterwards, each caecum or colon was incubated in calcium- and magnesium-free HBSS containing 2.5% fetal bovine serum and 1 mm DTT for 20 min at 37° to remove the mucus, washed three times and then incubated twice in calcium- and magnesium-free HBSS containing 2.5% fetal bovine serum and 1 mm EDTA for 20 min at 37° with one wash between the two incubations. Following the second incubation, the samples were washed three times. The tissues were then incubated in calcium- and magnesium-free HBSS containing 2.5% fetal bovine serum, 400 U/ml collagenase type 3 (Worthington Biochemical, Freehold, NJ) and 0.5 mg/ml DNase I (Roche, Indianapolis, IN) for 90 min at 37°.
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