Q: What is the assay principle for your ECM Cell Adhesion Assays?
A: Our Cell Adhesion Assays use an ECM coated plate for cell seeding. The wells are then washed with PBS to remove any non-adherent cells. The remaining adherent cells are stained and the dye is then extracted for quantification using a plate reader.
Q: What is the origin of the matrix proteins?
A: The ECM proteins coated on the plate consist of mouse laminin, bovine collagen I, human fibronectin, human collagen IV, and/or human fibrinogen.
Q: Why is BSA used as a negative control?
A: BSA is a negative control because cell adhesion to ECM is mediated through integrin receptors and cells will not attach to BSA-coated wells.
Q: Does the plate need to be used all at once?
A: Our 48-well Cell Adhesion Assay is not provided as a strip well plate, but it is possible to save any unused wells for subsequent experiments as long as the solutions do not spill over into the unused wells. It is recommended to expose the plate to a UV light under the hood before re-using.
Q: What confluency is recommended?
A: We recommend plating cells to 50-75% confluency for the cell adhesion assay.
Q: Why does the protocol require serum free media?
A: It is important to use serum free media with the adhesion assay because serum contains many ECM proteins, which could potentially coat the wells of the plate. If this were to happen, it would complicate the results and make it impossible to know if the cells were binding to the specific protein coated on the plate or one of the many ECM proteins contained in the serum. Although most cell lines prefer 10% FBS for growth, they are typically fine during the short incubation time without FBS during this assay.
Q: Is this kit compatible with cells that are a different species than the ECM proteins?
A: Adhesion of cells to ECM proteins does not depend on species compatibly or by how well the proteins are conserved between species. Rather, adhesion depends on whether the cell expresses the integrins required for attachment. For example, if mouse cells express the integrin receptor for fibronectin, the cells would be able to bind to both human and mouse fibronectin. To know if this assay is compatible with mouse cells, it would be necessary to determine if the cell line being used expresses integrin receptors for these ECM proteins.
Q: Will using trypsin when preparing the cell suspension cleave the cell surface receptors?
A: We always use tryspin/EDTA to detach cells when preparing cell suspensions for our cell adhesion assay. The cell adhesion receptors recycle fairly fast, even after detachment by trypsin/EDTA. If you think trypsin/EDTA is too harsh for your cell type, you can consider using Versene (an EDTA solution for use as a gentle non-enzymatic cell dissociation reagent, formulated as 0.2 g EDTA(Na4) per liter of Phosphate Buffered Saline (PBS)).
Q: What cell line can be used as a positive control?
A: We have tested these assays with MDA-MB-231, HT-1080, and HEK293 cells, with MDA-MB-231 being the most adherent cells on each ECM protein. These cells would be a good positive control for this assay. Please see Figure 1 in the product manual for #CBA-070.