By Sean P. Colgan

A flexible number of without problems reproducible cell-cell interplay assays for uncovering mobile interactions on the molecular point, either in vitro and in vivo. The protocols hide a various set of cell-cell interplay versions in either basic and pathological states, are comfortably adaptable to just about any mobilephone sort and organ procedure, and comprise fundamental info and end result research. furthermore, the protocols stick to the profitable tools in Molecular Biology™ sequence structure, each one supplying step by step laboratory directions, an creation outlining the rules at the back of the procedure, lists of the required gear and reagents, and pointers on troubleshooting and averting identified pitfalls.

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Extra info for Cell-Cell Interactions: Methods and Protocols (Methods in Molecular Biology Vol 341)

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This permits a three- or four-color analysis of the population structure within the same microscopic field. The hybridizations with different probe combinations are cumulatively extended until the position of all relevant bacterial groups to each other and their relative concentrations are clarified. When probes for unrelated bacterial groups hybridize with the same bacteria, it is necessary to adjust the hybridization stringency (see Note 4) until a clear differentiation of the bacterial groups is possible.

Ice-cold methanol-acetone 1Ϻ1 (v/v) for fixing cells. 5% (w/v) Crystal violet (Sigma), 20% methanol, 80% dd H2O. Destaining solution: acetic acid/methanol/dd H2O (10/30/60, v/v/v). Tissue culture 96-well plates (Greiner). Microplate reader (BIO-TEK, Neufahrn, Germany). 8. THP-1 Cell Transendothelial Migration 1. 5-mm Transwells with a 8-µm pore size (Costar, Bodenheim, Germany). 2. 2% gelatin-precoated transwells. 3. 1% BSA. 4. Antibodies that were used in the adhesion assay: MAb LPM19c against Mac-1; MAb L15 against LFA-1; MAb Gi18 against platelet endothelial cell adhesion molecule-1 (PECAM-1) (kindly provided by Dr.

5, 40 mg/L penicillin, 8 mg/L ampicillin, 90 mg/L streptomycin (all Sigma); and 5% newborn calf serum (Sigma). 2. 25%) and EDTA (1 mM) from Invitrogen. 3. Hank’s Balanced Salts Solution (HBSS) buffers. 4). HBSS without Ca2+ and Mg2+ is termed HBSS– (for 1 liter: 990 mL of deionized-distilled Interactions of Intestinal Epithelial Cells 23 5. , Cambridge, MA). 6. Triton X-100 (10% solution; Sigma). 7. N-formyl-Met-Leu-Phe (1 µM stock solution; Sigma). 2. Solution of Rat Tail Collagen 1. 2. 3. 4. Rat tails.

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