C57BL/6J wild-type (wt) mice were obtained from the Jackson Laboratories and maintained under pathogen-free conditions in the animal facility of the Medical Faculty Carl Gustav Carus. The experiments were performed under approval by the Animal Welfare Committee of the State of Saxony (permission number 24-9168.24-1/2010/25). Mice were used for tissue removal only under euthanasia conditions approved by the Animal Welfare Committee of the Technische Universität Dresden. Euthanasia was performed using carbon dioxide anesthesia. Splenic B cells were isolated from 8- to 10-week-old C57BL/6J mice. The spleen was dissected from the mouse, meshed on a 40 µm nylon mesh and resuspended in culture medium Hybridoma-SFM (12045-084, InVitrogen), supplemented with 10% FCS, 100 U/ml penicillin, 100 U/ml streptomycin and 5×10−5 M ß-mercaptoethanol. B cell cultures were prepared using B cells purified on MACS columns according to the manufacturers instructions (130-090-862, Miltenyi Biotec). For the switch to IgG1, purified B cells were cultured at a cell density of 3×105 cells/ml in complete culture medium supplemented with 50 µg/ml LPS (L6511, Sigma) and 2 ng/ml mouse recombinant IL-4 (404ML, R&D Systems).
L6511
Manufactured by Merck Group
The L6511 is a piece of laboratory equipment manufactured by Merck Group. It is designed to perform a specific function within the laboratory setting. The core function of the L6511 is to provide a controlled environment for various scientific experiments and analyses. However, without further details about the specific capabilities and intended use of this equipment, a more detailed description cannot be provided while maintaining an unbiased and purely factual approach.
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5 protocols using l6511
1
Isolation and Culture of Mouse Splenic B Cells
2
Investigating G-CSF Modulation in LPS-Induced Inflammation
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Mice received two i.p. injections of 30 µg of LPS from Salmonella enterica serotype typhimurium (L6511 Sigma-Aldrich) at 0 and 24 h, and the analysis was performed 24 h later. Plasma from non-treated or LPS-treated mice was collected, and G-CSF levels were determined using a Luminex Assay Kit (Thermo-Fisher). Some mice received daily i.p. injections of 6 µg of G-CSF (r-metHuG-CSF, Filgrastim; Amgen) for three consecutive days, and their cells were analysed 24 h later.
3
Sepsis Induction Model in Mice
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Treatment was performed as described by Jiménez-Alcázar et al. (2017) (link). Mice were treated with three daily intraperitoneal injections of 1 μg/g of LPS from Salmonella enterica serotype typhimurium (L6511; Sigma-Aldrich) diluted in sterile PBS. With the last dose, mice received an i.v. injection of 1.5 × 107 heat-killed E. coli/gm body weight. The shown survival time indicates the time after E. coli injection. Mice that reached 20% weight loss prior to E. coli injection were excluded from the survival analysis. Blood and organs were collected at the time of euthanasia. Mice were scored as “non-surviving” if the animals showed rare signs of severe distress (hunched posture, paralysis, inability to walk, irresponsiveness to touch, or inability to consume food or water) or if they reached 20% weight loss. All surviving mice were euthanized and scored as “surviving” 42 h after the i.v. injection of heat-killed E. coli. E. coli (ATCC25922) was grown overnight at 37°C with shaking in lysogeny broth media. Bacteria were pelleted by centrifuging at 4,000 g for 5 min, washed with PBS, and resuspended in PBS. Aliquots of 1.5 × 109 bacteria/ml were incubated at 70°C for 15 min to heat-kill the bacteria. Aliquots were stored at −20°C until further use.
4
Evaluating Inflammatory Responses in Zebrafish
5
Salmonella Infection Model in Mice
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Purified LPS extract from S. typhimurium (Sigma, L6511) was dissolved in sterile phosphate-buffered saline (PBS) at 1 mg/ml and frozen in aliquots at −20°C. Animals were administered LPS via intraperitoneal injection at 10 mg/kg according to previous studies (15 (link)–17 (link)). Animals were infected with 5 × 106 colony-forming units (CFU) of S. typhimurium via intraperitoneal injection.
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