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Plate Treatments, Coatings and Applications

Plates for Cell-based Assays


Tissue-culture treated plates

Polystyrene plastic must be subjected to a surface treatment to render the plastic suitable for cell attachment. Untreated polystyrene surfaces are not suitable for cell attachment due to the surface chemistry of the polystyrene. The tissue culture treatment process involves exposing a polystyrene microplate to a plasma gas in order to modify the hydrophobic plastic surface to make it more hydrophilic. The resulting surface carries a net negative charge due to the presence of oxygen-containing functional groups such as hydroxyl and carboxyl. In general, this will lead to increased cell attachment.

Poly-lysine-coated plates

Poly-lysine is a synthetic positively-charged polymer, existing as two enantiomers: Poly-D-lysine (PDL) and Poly-L-lysine (PLL). Adherence of certain cell types to poly-lysine-coated surfaces is based on the electrostatic interaction of the poly-D-lysine polycation with the negative charges of the cell membrane. Use of poly-lysine coatings on plate surfaces can help mediate the negative charges of the cell membrane and the negative charge of the surface. Both PDL and PLL are commonly used however PDL is not degraded by cellular proteases and is therefore often the preferred choice. As Poly-lysine is a synthetic protein, it does not influence the signaling pathways of the cells and is completely free of any animal contaminants. Almost all cell types will adhere to Poly-lysine coated plate bottoms.

Collagen-coated plates

Extracellular matrix proteins such as collagen provide an attachment framework for the adhesion and growth of certain cell types in vivo, and can also be used for cell attachment to plate surfaces in vitro. Cellular fibronectin membrane proteins mediate the attachment of cells to collagen substrates. Collagen is the most abundant protein in mammals that is found throughout the body and is a major component of the extracellular matrix (ECM). The most frequently used types of collagen for coating are collagen I and IV. Collagen type I is suitable for endothelial and epithelial cells, muscle cells and hepatocytes. Collagen type IV is the major constituent of basement membranes and offers more physiologically relevant conditions to cells as well as improving the adherence of specific cell types i.e. PC-12 (rat adrenal pheochromocytoma cell line). Our catalog plates are coated with rat tail collagen Type I.

Sterility

All tissue-culture treated plates are sterilized using gamma irradiation to prevent contamination.


References on treatments and coatings for cell-based assays

  1. Harnett EM, Alderman J, Wood T. The surface energy of various biomaterials coated with adhesion molecules used in cell culture. Colloids and Surfaces B: Biointerfaces 2007 Mar;55(1):90-97.
  2. Barker SL, LaRocca PJ. Method of production and control of a commercial tissue culture surface. Journal of Tissue Culture Methods 1994 Sep;16(3-4):151-153.
  3. Hayman EG, Pierschbacher MD, Ruoslahti E. Detachment of cells from culture substrate by soluble fibronectin peptides. The Journal of Cell Biology 1985 Jun;100(6):1948 -1954.
  4. Curtis AS, Forrester JV, McInnes C, Lawrie F. Adhesion of cells to polystyrene surfaces. J. Cell Biol 1983 Nov;97(5 Pt 1):1500-1506.
  5. Knox P, Wells P. Cell adhesion and proteoglycans. I. The effect of exogenous proteoglycans on the attachment of chick embryo fibroblasts to tissue culture plastic and collagen. J. Cell. Sci 1979 Dec;40:77-88.
  6. Wicha MS, Liotta LA, Garbisa S, Kidwell WR. Basement membrane collagen requirements for attachment and growth of mammary epithelium. Experimental Cell Research 1979 Nov;124(1):181-190.
  7. Hahn LH, Yamada KM. Identification and isolation of a collagen-binding fragment of the adhesive glycoprotein fibronectin. Proc. Natl. Acad. Sci. U.S.A 1979 Mar;76(3):1160-1163.
  8. Murray JC, Stingl G, Kleinman HK, Martin GR, Katz SI. Epidermal cells adhere preferentially to type IV (basement membrane) collagen. The Journal of Cell Biology 1979 Jan;80(1):197 -202.
  9. Yavin E, Yavin Z. Attachment and culture of dissociated cells from rat embryo cerebral hemispheres on polylysine-coated surface. J. Cell Biol 1974 Aug;62(2):540-546.

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Coating techniques


Passive coating vs. covalent coating

Proteins and small molecules can be coated either passively or covalently to the plate. Passive coating creates a weaker association, compared to covalent coating (which creates a chemical bond between the streptavidin and the plate).

PerkinElmer plates that have covalently bound coatings (chemical bond):

  • Some streptavidin plate products
  • Nickel chelate
  • Protein A
  • WGA

 PerkinElmer plates that are passively coated (weaker bond)

  • Phospholipids
  • Some streptavidin plate products 
  • Antibodies
  • PDL (poly-D-lysine)
  • COL (collagen)

Binding capacity will also vary with plate-coating technique. Refer to the table below for an example:

Coating procedureWell format Binding capacity
Covalent  9640 pmole biotin/well
 38412 pmole biotin/well
Passive 

 

 9610 pmole biotin/well
 384 

2-4 pmole biotin/well

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Plates for biochemical assays


Polyethyleneimine (PEI)-coated plates

Polyethyleneimine (PEI) is a cationic polymer that can neutralize the negative charge of glass fiber filters (GF/C, GF/B). Treating GF/C and GF/B filter plates with polyethylenimine (PEI) is often used to minimize non-specific binding, particularly in ligand-binding assays. For some applications involving negatively-charged ligands (such as GTP binding assays), PEI coating could cause problems by creating a positively-charged surface that actually promotes non-specific binding to the filter, due to the negative charge on the ligand.

PEI coating is also sometimes used to coat the surface of glass plates, to neutralize the negative charge of the glass. This can help facilitate cell attachment in certain situations.

 

Wheat germ agglutinin (WGA)-coated plates

Wheat germ agglutinin is commonly used to anchor cell membranes to the surface of a plate. Wheat germ agglutinin can interact with cell-surface sugars/glycoproteins.

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Protein-coated plates


Streptavidin-coated plates

Streptavidin-coated plates are often used to create generic plates for solid-phase (coated plate) assays, such as ELISA assays, DELFIA® immunoassays, and FlashPlate® assays. Streptavidin will bind biotinylated antibodies, biotinylated proteins, and other biotinylated moieties, anchoring the biotinylated reagent to the well of the plate. 

 

Antibody-coated plates

Antibody-coated plates are also frequently used to create plates for solid-phase (coated plate) assays. Some of our DELFIA plates for DELFIA fluorescence assays as well as some of our FlashPlate microplates are offered pre-coated with anti-species antibodies, to be used to anchor a relevant antibody to the plate. Additionally, we can custom coat your antibody to our plates if desired.

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Custom plate services at PerkinElmer


PerkinElmer offers custom microplate services, including bulk ordering, fast and flexible plate barcoding, biological plate coating (including poly-D-lysine, collagen, streptavidin coating, antibody coating, and other coatings on request), custom tissue culture-treatment, custom high protein binding treatment, custom sterilization of microplates, special packaging, and other microplate treatments. If you are interested in custom plate services, please contact our custom service team:

ON>POINT® Custom Services

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