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By John R. Gordon

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METHOD 7 Expression of the FcεRI is inducible in mast cells, and this is at least in part regulated the concentrations of exogenous IgE, such that in the presence of high concentrations of IgE, mast cells express more IgE receptors. Thus, freshly purified tissue mast cells, which will likely have the vast majority of their existing FcεRI occupied by IgE antibodies of irrelevant specificities, can be best sensitized with IgE of the desired specificity by incubating the cells overnight in high concentrations of the antibody.

1 100 TIB211 IgM (45% AS) 2. 3. 4. 5. 6. 5, or TIB211 preparations) to each tube. Incubate the cells with the antibody for 30 min at room temperature. Add 100 µl of the guinea pig complement to each tube, and incubate the tubes at 37˙C for 30 min. Wash the cells two times with DMEM-10% FCS (8 min at 1500 rpm in the clinical centrifuge). Resuspend the cells to 100 µl in DMEM-10% FCS, and add 3 µl of FITC-labelled anti-CD4 and 3 µl of PE-labelled anti-CD8 IgG to each tube and incubate the cells on ice for 30 min.

5 µg/ml. 5x106 cells/well. , total well volume of 200 µl). Place the plates in the 37˙C CO2 incubator for three days. 49 3. 4. 5. At the end of the three days, examine the cells in each well to get a feeling for how the cells have responded to the ConA (strong ConA mitogenic responses will have induced cell clumping). Next, add 20 µl of MTT solution (5 mg/ml) to each well and return the plates to the incubator for 45 - 90 min. Remove 150 µl of medium from each well, taking care not to also aspirate cells from the bottom of the wells.

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