AIM: performing a cell adhesion assay using a 96-well plate coated with laminin-1 or

fibronectin
Introduction:
A cell adhesion assay is a laboratory technique used to assess the ability of cells to adhere to
a substrate or other cells. It provides quantitative or qualitative measurements of cell
adhesion and is valuable for studying cellular interactions, migration, and the effects of
various factors on cell adhesion.
Cell adhesion refers to the process by which cells attach to and interact with other cells or the
extracellular matrix (ECM). It plays a crucial role in various biological processes, including
tissue development, wound healing, immune response, and the formation of multicellular
structures.
There are different types of cell adhesion mechanisms that cells employ to bind to one
another or to the ECM:
1. Cell-Cell Adhesion: a. Tight Junctions: Found in epithelial tissues, tight junctions form
a seal between adjacent cells, preventing the passage of molecules between them. b.
Adherens Junctions: These junctions connect adjacent cells through transmembrane
proteins called cadherins, which link to the actin cytoskeleton inside the cells. c.
Desmosomes: Desmosomes provide strong adhesion between cells through
intermediate filaments, such as keratin, which extend across the cytoplasm of the cells.
d. Gap Junctions: Gap junctions facilitate direct communication between cells by
forming channels that allow the passage of small molecules and ions.
2. Cell-ECM Adhesion: a. Integrins: Integrins are transmembrane proteins that link the
cell's cytoskeleton to the ECM components, such as fibronectin, collagen, and laminin.
b. Focal Adhesions: Focal adhesions are multiprotein complexes formed at the cell-
ECM interface, connecting integrins to the actin cytoskeleton. c. Hemidesmosomes:
Hemidesmosomes anchor epithelial cells to the basement membrane by linking
integrins to intermediate filaments, such as keratin.
Materials to be prepared:
1. Washing Buffer: 0.1% BSA in medium (DMEM or RPMI)
2. Blocking Buffer: 0.5% BSA in medium (DMEM or RPMI)
3. Laminin-1 10-12 μg/ml or FN 20 μg/ml
4. 96-well-plate
5. Crystal violet (5mg/ml in 2% ethanol)
6. 1% SDS in H2O
7. 4% paraformaldehyde
Procedures:
 1. Coat 96-well-plate with Laminin-1 or FN at 37 oC for 1 hr or at 4 oC O/N. Leave some
wells uncoated as negative control.
2. Wash with washing buffer for 2 times.
3. Block plates with blocking buffer at 37 oC in CO2 incubator for 45-60 minutes.
4. Wash with washing buffer.
5. Chill the plates on ice.
6. Count cell to 4 X 105/ml. Add 50 μl cells in each well.
7. Incubate in CO2 incubator at 37 oC for 30 minutes.
8. Shake the plate at 2000 rpm for 10-15 seconds. Wash with washing buffer 2-3 times.
9. Fix with 4% paraformaldehyde. Incubate at RT for 10-15 minutes.
10.Wash with washing buffer.
11.Stain with Crystal Violet for 10 minutes.
12.Wash with water.
13.Turn the plates upside down. Let the plates dry up completely.
14.Add 2% SDS. Incubate at RT for 30 min.
15.Read plate at 550μm.
Explinstion:
Laminin-1 is a specific isoform of laminin, which is an important component of the
extracellular matrix (ECM). Laminin-1 plays a significant role in cell adhesion, migration,
and tissue development. It is often used in cell adhesion assays to promote and study cell
attachment and spreading. Here's how laminin-1 can be used in a cell adhesion assay:
1. Coating the Substrate: a. Prepare a solution of laminin-1 at the desired concentration
in a suitable buffer (e.g., phosphate-buffered saline, PBS). b. Add the laminin-1
solution to a culture plate or other suitable substrate (e.g., glass coverslips) to coat the
surface evenly. c. Incubate the coated substrate at an appropriate temperature (usually
37°C) for a specific duration (typically 1-2 hours) to allow laminin-1 to adsorb and
form a coating layer.
2. Cell Seeding and Adhesion: a. Prepare the cells of interest in a suspension or single-
cell suspension by detaching them from culture plates or tissues using standard
methods. b. Collect the desired number of cells and resuspend them in a suitable cell
culture medium or buffer. c. Carefully add the cell suspension onto the laminin-1-
coated substrate, ensuring uniform distribution across the surface. d. Incubate the cells
at the appropriate conditions (e.g., temperature, humidity, CO2 levels) to allow for
adhesion and attachment to the laminin-1-coated surface. The incubation time can
vary depending on the cell type and experimental requirements.
 3. Gentle Washing: a. After the incubation period, gently wash the substrate to remove
any non-adherent cells or debris. b. Use a suitable buffer (e.g., PBS) and perform the
washing steps with care to avoid disrupting the adherent cells. c. Repeat the washing
steps if necessary to ensure that only firmly adhered cells remain on the laminin-1-
coated surface.
observations and results you can expect from this protocol:

 Coating the Plate:

 The laminin-1 or FN coating provides a substrate for cell adhesion. Wells without
coating serve as negative controls to compare against.
 Washing and Blocking:

 Washing the plate removes any unbound laminin-1 or FN and other potential
contaminants.
 Blocking with a blocking buffer helps prevent non-specific binding of cells to the well
surface.
 Cell Seeding:

 The chilled plate helps promote cell attachment.

 Adding 50 μl of cells to each well allows cells to adhere to the coated surface.
 Incubating the plate at 37°C for 30 minutes provides sufficient time for cells to attach
and spread.
 Washing and Fixation:

 Shaking the plate and washing remove non-adherent cells, leaving only the adherent
ones.
 Fixation with 4% paraformaldehyde preserves the cell morphology and adherence
state.
 Staining and Drying:

 Staining with Crystal Violet allows visualization and quantification of adherent cells.
Adherent cells will be stained and can be observed as visible clusters or monolayers.
 Inverting the plate and allowing it to dry completely ensures that excess staining
solution is removed.
 SDS Treatment:
  Addition of 2% SDS solubilizes the stained cells, releasing the dye into the solution.
This step is necessary for the subsequent absorbance measurement.
 Reading:

 The absorbance at 550 nm is measured using a plate reader.

 The absorbance value correlates with the number of adherent cells or the extent of cell
adhesion. Higher absorbance indicates more cells adhering to the coated surface.
results:
1. Observation:
 Upon visual inspection, you observe that some wells have a distinct monolayer
of adherent cells, while others may have fewer cells or show no visible
adhesion.
2. Staining:
 When you stain the cells with Crystal Violet, the wells with a higher number of
adherent cells will exhibit a more intense purple staining. This indicates a
greater extent of cell adhesion.
 Conversely, wells with fewer adherent cells or no adhesion will appear lighter in
color or may show minimal staining.
3. Absorbance Measurement:
 After solubilizing the stained cells with 2% SDS, you measure the absorbance at
550 nm using a plate reader.
 The wells with a higher number of adherent cells will yield higher absorbance
values. These wells indicate a stronger cell adhesion to the laminin-1-coated
surface.
 In contrast, wells with fewer adherent cells or no adhesion will result in lower
absorbance values, indicating weaker or no cell attachment.
4. Data Analysis:
 By comparing the absorbance values of the laminin-1 coated wells to the
negative control wells (uncoated), you can determine the specific effect of
laminin-1 on cell adhesion.
 If the absorbance values of the laminin-1 coated wells are significantly higher
than the negative control wells, it suggests that laminin-1 promotes cell
adhesion for the tested cell type.
 Conversely, if the absorbance values of the laminin-1 coated wells are similar to
or not significantly different from the negative control wells, it indicates that
laminin-1 may not have a substantial effect on cell adhesion in this context.