Sera from mice in each treatment group were examined for neutralization activities against STa and CT enterotoxicity using T-84 cells (CCL-248, ATCC) and a cyclic GMP or AMP EIA kit (Enzo Life Sciences, Farmingdale, NY, USA). As we described previously (24 (link), 25 (link), 28 (link), 29 (link)), 30 µL mouse serum sample pooled from each group was mixed with 2 ng STa toxin (BEI Resources, ATCC) or 30 ng CT (Sigma; we increased CT from 10 ng to 30 ng in an attempt to differentiate neutralization activity against CT from antibodies from the vaccine antigen versus dmLT adjuvant) and incubated at room temperature for 30 min and then transferred to a well of a 24-well tissue culture plate (Falcon) containing 95%–100% confluent monolayered T-84 cells and incubated in a CO2 incubator for 1 h (for cGMP) or 3 h (for cAMP). Cells were rinsed thoroughly with PBS to remove extracellular cGMP or cAMP, then dislodged, and lysed with 0.5% Triton to release intracellular cGMP or cAMP. The intracellular cGMP or cAMP levels (nM, picomole per mL) were measured by following the manufacturer’s protocol (Enzo Life Science). T-84 cells in culture medium without treatment with mouse sera or toxin were also included in the assay to measure intracellular cGMP or cAMP baseline levels.
T 84 cells ccl 248
Manufactured by American Type Culture Collection
Sourced in United States
T-84 cells (CCL-248) are a continuous cell line derived from human colorectal adenocarcinoma tissue. The cells exhibit an epithelial morphology and are used in research applications.
Automatically generated - may contain errors
2 protocols using t 84 cells ccl 248
1
Neutralization of Enterotoxins by Mouse Sera
2
Epithelial Cell Priming and Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Where indicated, cells were primed with 100ng/ml or 400ng/ml of Pam3CSK4 (Invivogen) for 3 hours prior to infection. To induce SPI-1 expression, overnight cultures of Salmonella were diluted into LB broth containing 300 mM NaCl and grown for 3 hours standing at 37°C (28) . Overnight cultures of Listeria were diluted and grown shaking for Cell culture of intestinal epithelial cell lines All cell lines were obtained from American Type Culture Collection (ATCC).
Caco-2 cells (HTB-37; ATCC) were maintained in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. T84 cells (CCL-248; ATCC) were maintained in DMEM F-12 supplemented with 5% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin.
One day prior to infection or treatment, cells were dissociated with 0.25% Trypsin-EDTA (Gibco) diluted 1:1 with 1X PBS. Cells were incubated with trypsin at 37°C for 15 minutes, after which the trypsin was neutralized with serum-containing media. Cells were replated in media without antibiotics in a 24-well plate at a concentration of 3 × 10 5 cells/well. Where indicated, cells were primed with 100ng/mL or 400ng/ml Pam3CSK4 (Invivogen) or 500ng/ml LPS (Sigma-Aldrich) for 3h or 16h prior to anthrax toxin treatment or bacterial infections.
Caco-2 cells (HTB-37; ATCC) were maintained in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. T84 cells (CCL-248; ATCC) were maintained in DMEM F-12 supplemented with 5% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin.
One day prior to infection or treatment, cells were dissociated with 0.25% Trypsin-EDTA (Gibco) diluted 1:1 with 1X PBS. Cells were incubated with trypsin at 37°C for 15 minutes, after which the trypsin was neutralized with serum-containing media. Cells were replated in media without antibiotics in a 24-well plate at a concentration of 3 × 10 5 cells/well. Where indicated, cells were primed with 100ng/mL or 400ng/ml Pam3CSK4 (Invivogen) or 500ng/ml LPS (Sigma-Aldrich) for 3h or 16h prior to anthrax toxin treatment or bacterial infections.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!