Cr strain, DBS100 (ATCC) was used for scRNA-seq experiments. For flow cytometry analysis and to track Crin situ, we used a strain of Cr expressing GFP61 (link) (derived from DBS100; provided by B. A. Vallance). For whole-body imaging experiments, we used the bioluminescent Cr strain ICC18062 (derived from DBS100; generously provided by G. Frankel and S. Wiles). A fresh, single colony was grown in 10 ml LB at 37 °C with rotation at 225 rpm for 12–14 h. Next day, 1 ml of overnight culture was added to 250 ml LB broth, incubated at 37 °C with rotation (225 rpm) for 3–3.5 h until OD600 reached 1.0 on spectrophotometer. Bacteria were pelleted at 25 °C, 3,000 rpm for 15 min and then resuspended in 5 ml sterile PBS. Mice were inoculated with 2 × 109 CFU in a total volume of 100 µl of PBS by gastric gavage.
Dbs100
Manufactured by American Type Culture Collection
Sourced in United States
The DBS100 is a digital bioindicator system designed for monitoring and validating sterilization processes. It provides real-time data on the efficacy of sterilization methods, enabling users to ensure the safety and quality of their procedures.
Automatically generated - may contain errors
Lab products found in correlation
Macconkey agar, by HiMedia (3 mentions)
Brilliance e coli coliform selective agar, by Thermo Fisher Scientific (3 mentions)
Chloramphenicol, by Merck Group (2 mentions)
Macconkey agar plate, by Solarbio (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Lysing matrix d, by MP Biomedicals (2 mentions)
Mini beadbeater 8, by Biospec (2 mentions)
5 mm metal bead, by Qiagen (2 mentions)
Monoclonal anti il 22 clone 8e11, by Genentech (2 mentions)
Il 22 fc pro312045, by Genentech (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
2 3 cgamp, by InvivoGen (1 mentions)
Recombinant gm csf, by BioLegend (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
B6 cg mir34atm1lhe j, by Jackson ImmunoResearch (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
Lps e coli o111 b4, by Merck Group (1 mentions)
60 protocols using dbs100
1
Bacterial Inoculation via Gastric Gavage
2
C. rodentium Infection Model in Mice
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Sex matched, 8 to 10 week old mice, fasting for 8 hr, were orally gavaged with 1.2 × 1010 cfu of C. rodentium strain DBS100 (ATCC 51459). In certain instances, mice were intraperitoneally injected with 200 μg of murine LTβR-IgG1 fusion protein or MOPC21 isotype control (Biogen Idec) 1 day prior to infection, as described (Wang et al., 2010 (link)). IECs, isolated following published procedure (Greten et al., 2004 (link)), were utilized for biochemical analyses. For histology, dissected colons were fixed in 10% neutral buffered formalin. Paraffin-embedded tissue sections were stained with anti-myeloperoxidase antibody (Pierce, Waltham, Massachusetts, USA) for neutrophil recruitment or with Hematoxylin and Eosin (H&E) for tissue pathology evaluation. Fecal samples were weighed, homogenized, and serially diluted homogenates were plated on MacConkey agar (HiMedia, Mumbai, India) to score for C. rodentium. Similarly, spleens and livers were aseptically removed and assessed for bacterial load. For bone marrow chimera experiment, recipient WT or Nfkb2−/− mice were lethally irradiated and marrow cells from the indicated donor mice were transferred. After 6–8 weeks, mice were infected.
3
Generating Murine Dendritic Cells
4
Acute Intestinal Inflammation Induction with GFP-C. rodentium
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Green fluorescent protein-labeled C. rodentium DBS100 (ATCC 51459) was used to incite acute inflammation. The bacterium was grown aerobically on lysogeny broth agar (LA) with 30 µg/ml chloramphenicol at 37°C for 24 h. To differentiate GFP-labeled C. rodentium from nonlabeled C. rodentium, a chloramphenicol resistance gene was incorporated into the genome, which required chloramphenicol to be used for growth and isolation techniques. Biomass was removed from the surface of the agar and transferred into sterile lysogeny broth (LB) containing 15 µg/ml chloramphenicol (Sigma-Aldrich). Cultures were maintained for 2 h at 37°C at 100 rpm until an optical density at 600 nm (OD600) of >0.1 was obtained. Cultures were centrifuged at 2,256 × g for 15 min, supernatants were removed, and C. rodentium cells were resuspended in 3.0 ml PBS. To confirm densities of viable cells, inoculum was diluted in a 10-fold dilution series and 100 µl of each dilution was spread in duplicate onto LA. Cultures were incubated at 37°C, and the number of C. rodentium colonies was counted at the dilution yielding 30 to 300 CFU after 24 h. Cell densities were adjusted to 3 × 109 CFU/ml with PBS. For each replicate, 12 mice were administered C. rodentium cells and PBS (100 µl) or PBS alone (100 µl) via gavage on two consecutive days using a 22-gauge-by-2.5-cm-long gavage needle with a 1.25-mm ball tip.
5
Oral Infection of Mice with C. rodentium
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8–12-week-old age- and sex-matched mice were orally administered 1×1010 CFU C. rodentium (DBS100, ATCC). Body weight was measured until day 10. Feces were collected on days 8 and 10 for CFU counting. Briefly, feces were weighed, homogenized, and serially diluted up to 10−6 in sterile PBS. Three consecutive dilutions per sampled were plated on MacConkey agar plates and incubated at 37 degrees C. Colonies were then counted and counts for three dilutions were averaged and normalized to feces weight.
6
Bacterial Colitis Induction in Mice
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Mice were infected with 1 × 109 colony-forming units (CFUs)/mouse of the CR strain DBS 100 (ATCC 51459) to cause bacterial colitis. Briefly, the sterile Luria–Bertani medium was injected into a single CR colony grown on a fresh MacConkey agar plate (Solarbio, Beijing, China) and shaken overnight at 37 °C [21 (link)]. Mice were infected after three weeks of being fed a chow diet.
7
Isolation and Characterization of C. rodentium
8
Oral C. rodentium Infection in Mice
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8–10-wk-old age- and gender-matched mice were orally administered 2 × 109C. rodentium, strain DBS100 (ATCC), as previously described (Cella et al., 2009 (link)). Body weight of mice was measured until day 7. For Rapa injection, mice were injected i.p. with 200 µl of Rapa (5 mg/kg body weight) or DMSO vehicle before infection.
9
Citrobacter rodentium Mouse Model
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Citrobacter rodentium strain DBS100 was purchased from ATCC and used for all inoculations. 6–8-weeks old mice were orally gavaged with approximately 1–2 × 109 CFU in a total volume of 200 µl of PBS. To assess bacterial burdens, feces and tissues (first 0.5 Cm of distal colon and spleen) in PBS were homogenized with Lysing Matrix D (MP Biomedicals) using Precellys homogenizer. Serially diluted homogenates were then plated on MacConkey agar (HiMedia Laboratories). For histology scoring, the second 0.5 cm pieces of distal colon were fixed in 10% formalin, stained with hematoxylin and eosin, and scored as described previously (http://www.jove.com/video/50222/the-citrobacter-rodentium-mouse-model-studyingpathogen-host ) . To measure cytokine levels, the third 0.5 cm of distal colon was homogenized as described above in RLT lysis buffer of RNeasy Kit (Qiagen).
10
Mouse Infection with Salmonella and Citrobacter
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The naturally streptomycin-resistant wild type strain S. enterica serovar Typhimurium SL1344 [47] (link), a generous gift from Prof. Mikael Rhen was used in this study. Naturally occurring naldixic acid-resistant Citrobacter rodentium, DBS100 (ATCC 51459) was also used for mouse infection. Prior to inoculation into host mice, strain SL1344 and C. rodentium were grown overnight at 37°C in Luria-Bertani (LB) broth, diluted 1∶20 in fresh medium, and sub-cultured for 3–4 h under mild aeration until an optical density of 0.4 to 0.6 at 600 nm was reached. Bacteria were washed twice in cold PBS and then suspended in cold PBS for mouse inoculation.
The cecum and spleen from post-sacrifice mice were collected in 1 mL of sterile PBS. Samples were kept on ice, minced, and homogenized. Serial dilutions of the homogenates were plated on LB agar plates supplemented with 100 µg/mL streptomycin to enumerate S. Typhimurium. Plates were incubated overnight at 37°C, and colonies were counted thereafter.
The cecum and spleen from post-sacrifice mice were collected in 1 mL of sterile PBS. Samples were kept on ice, minced, and homogenized. Serial dilutions of the homogenates were plated on LB agar plates supplemented with 100 µg/mL streptomycin to enumerate S. Typhimurium. Plates were incubated overnight at 37°C, and colonies were counted thereafter.
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