Human Collagen Type IV

Cell adhesion plays a major role in cellular communication and regulation, and is of fundamental importance in the development and maintenance of tissues. Scientists are continually examining the adhesion and migration of many diverse cell types on various extracellular matrix (ECM) component proteins. Millicoat Cell Adhesion Strips are provided as 12 x 8-well removable strips in a plate frame for convenience and flexibility in designing assays. The wells in rows A - G have been coated with Human Collagen Type IV. Row H of each strip is coated with BSA which serves as a negative assay control. Cells are seeded onto the coated substrate. Subsequently, adherent cells are fixed and stained. Relative attachment is determined using absorbance readings.

PROCEDURE:

NOTE: Optimal assay timing and performance may vary for different cell lines but generally can be obtained using subconfluent cell cultures in the assay described below. Subconfluent cultures can be achieved by splitting cells 1 to 2 days prior to performing the assay.

1. Rehydrate the strips with 200 mL of PBS per well for at least 15 minutes at room temperature. Remove the PBS from the rehydrated strips.

2. Prepare a single cell suspension, preferably using a non-enzymatic dissociation buffer. Optimum cell density may be determined by titration of the cells. A common starting range is between 1x10E05 to 1x10E07 cells/mL.

3. Add 100 mL of the diluted cell suspension to each well. Incubate the plate at 37°C for 45 minutes in a CO2 incubator. Gently wash the plate 2-3 times with PBS containing Ca2+/Mg2+ (200 mL/well).

4. Add 100 mL of 0.2% crystal violet in 10% ethanol to each well. Incubate for 5 minutes at room temperature. Remove the stain from the wells. Gently wash the strips 3-5 times with PBS (300 mL/well) to remove the excess stain.

5. Add 100 mL of Solubilization Buffer (A 50/50 mixture of 0.1M NaH2PO4, pH 4.5 and 50% ethanol) to each well. Allow strips to incubate and gently shake at room temperature until the cell-bound stain is completely solubilized; approximately 5 minutes.

6. Determine the absorbance at 540 - 570 nm on a microplate reader.

Research Category
Cell Structure

96 wells

Strips may be stored at 2-8°C in the foil pouch for at least 3 months. Unused strips may be placed back in the pouch for storage. Ensure that the desiccant remains in the pouch, and that the pouch is securely closed.

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

MILLICOAT is a registered trademark of Merck KGaA, Darmstadt, Germany

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.