Oglethorpe
University
BIO 316 CELL BIOLOGY Fall 2008 Dr. Schmeichel
Figure 1. The transwell migration assay set up. Monolayers
of serum-starved adherent cells are trypsynized and cell suspensions are placed in serum-free medium into the upper
well of a transwell filter apparatus (blue). The filter is
suspended in a well of a 24-well plate and the lower
reservoir (purple) is filled with the same media (no cells)
plus an added chemoattractant (e.g., growth factor). The
cells are incubated under normal conditions for 2-24 hours.
Migration assays are terminated by retrieving the filter,
rubbing off non-migrated cells from top surface, and counting
cells that are found on the underside of the filter. (Adapted from
www.biocat.de/cgibin/adm/sub2.pl?sub1=cell_migration&sub2=colorome
tric_cell_migration_assays&main_group=cell_biology) Lab #4: Transwell Cell Migration Assays 10/28/08
Goals: · To assay the migratory behavior of MDCK cells in the
context of a transwell migration assay using H1299 lung
cancer cells as controls
· To learn how to count live cells in preparation for their use in a cell-based behavioral assay
· Investigate the role of HGF in eliciting increased motility due to epithelial to mesenchymal transition.
· Understand the difference between measuring migration and invasion and the role of Matrigel as a substitute
basement membrane
· Using the microscope to quantitate cell migration
· Express results of migration assay in graphical form using the graphing functions of Excel
Introduction:
modelsim
Cell migration, the movement of cells from one area to another generally in response to a chemical signal, is central to achieving functions such as wound repair, cell differentiation,
embryonic development and the
metastasis of tumors. The availability
电话银行是什么of methods to study cell migration is of great importance to allow a better
understanding of the underlying
biological mechanisms. Moreover,
since the correlation between the in
vitro migratory potential of tumor cells
and their in vivo invasive properties
was reported in 1998 (1), cell
migration assays have gained widespread acceptance in the screening of anti-cancer drugs. A broad spectrum of assays to analyze chemokinetic and chemotactic cell migration has been developed in previous years. Among them, the filter assay is one of the most frequently used in vitro assays. Firs
t published in 1962 by Stephen除湿胃苓汤
Boyden (2), the Filter assay is still
Figure 2. MHCC97-H hepatocellular employed in cell migration studies. This assay involved a two-compartment system where cells may be induced to migrate from an upper compartment through a porous membrane into a lower compartment, thus following the gradient of a chemotattractant (Figure 1). Relative to the size of the investigated cells, the size of the pores in the membrane (see magnified view of pores in Figure 2) must be small enough to avoid the passive passage of cells, but large enough to allow their active migration. Customarily, 3 um pores are Once the transwell migration assay is set up, the culture apparatus is incubated for a
period of time under 24 hours. To
determine the extent to which cells have
migrated, the filter inserts are removed from the culture wells, the cells that have not
migrated are physically cleared from the top
surface of the filter, and the remaining cells (i.e., those that have migrated to the underside of the filter) are fixed and stained. The migratory behavior of the cells are then quantitated by visual inspection of the filter membranes such that by viewing multiple fields, an area corresponding to 20% of the filter are counted. For these types of experiments it is always important to run each condition in multiple wells to improve the statistical significance of an observed
similarities and differences. (Today we will do 2 wells/ treatment – it’s always best to do 3!) Cell invasion is similar to cell migration however in vivo , invasion is characterized by the ability of cells to traverse a specific extracellular matrix barrier. The matrix underlying an epithelial cell for example, is a laminin-rich layer that is specifically referred to as a basement membrane or a basal lamina. Invasion through a basement membrane requires that a cell secrete a number of proteolytic/degradative enzymes into the extracellular space to make a path through which a cell can migrate. Cell invasion is
exhibited by both normal cells in responses such as inflammation and by tumor cells in the process of metastasis, where acquisition of an invasive phenotype is the hallmark of malignant tumor cell. The invasive capacity of a particular cell may be assayed using a modified version of the Filter assay described in Fig. 1 whereby the porous filter is coated with a matrix preparation that approximates tis
sue like ECMs, such as the basement membrane. (In these assays the ECM that coats the filter will “plug up” the 8 um pores, such that penetration through the filter requires that the cells first burrow through the ECM.) One commercially available source of BM is called Matrigel. Matrigel is recovered from the matrix produced in mice bearing Engelbreth-Holmes Swarm (EHS) tumors and has been demonstrated to closely approximate the biochemical characteristics of laminin-rich basement membranes.
In last week’s journal club, we considered a series of experiments in which cell behavior of cells derived from a uveal melanoma tumor was examined in a complex three dimensional matrix. In the report by Maniotis et al (3.) we learned that cells can respond to 3-dimensional culture by forming physiologically relevant three-dimensional structures and gained insight into the intimate interplay between cells and their surrounding environment. Today we are interested in querying the migratory properties of MDCK cells and their ability to migrate and invade through a variety of physical barriers. We will use the Boyden chamber migration assay to explore the migratory properties of these cells in response to a chemotactic stimulus provided by 10% FBS or HGF-containing medium (i.e., the normal growth medium of the cells that contains an abundance of growth factors and cytokines). We will also compare the migratory activity of MDCK cells to that of human H1299 lung carcinoma cells.
We will establish the migratory potential of MDCK cells using 10% FBS medium as a chemoattractant. The extent to which migration/chemotaxis has occurred will be evident by comparing assays in the presence and absence of 10% FBS (in FBS-minus assays, the same medium that cells are suspended in will be added to the bottom chamber). Sometimes cells will exhibit a more robust migratory response if the surfaces on which they are migrating are coated with a thin layer (i.e., not thick enough to block the pores!)
希腊数学家丢番图of the ECM component, Collagen I. We will measure the migratory potential of MDCK cells exposed to a variety of chemoattractive cues. We will also query the invasive potential of MDCK cells by comparing migration assays performed on collagen I-coated filters, and filters coated with a thick pore-blocking, Matrigel barrier layer . Experimental Procedures: Cells:
A 90% confluent T-75 flask of MDCK cells will be available for use by all of the students.
A comparable flask of H1299 human lung cancer cells will also be available. 24 hours before the laboratory, the 10% FBS growth medium will be exchanged to “Serum-Free” medium (i.e., 1X EMEM + 0.5% BSA). This will in essence “prime” the cells to make them more responsive to the growth fact
cvrors in the chemotactic medium. We will trypsinize this flask using standard procedures and resuspend the final pellet in 2 mL of Serum-Free medium (SF-EMEM). To continue with the experiment it is critical that we estimate the amount of cells added into each assay. To do that we will count the cells using a “Hemocytometer”.
Counting Cells Using a Hemocytometer
A device used for determining the number of cells per unit volume of a suspension is called a hemocytometer (See Fig. 3 below). The hemocytometer was originally designed for performing blood cell counts.
Fig. 3. Diagram of the hemocytometer that we will be using to count cell numbers in our HEK 293 cell
To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid. The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the V-shaped wells with a pasteur or other type of pipet. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power.
It is essential to be extremely careful with higher power objectives, since the counting chamber is much thicker than a conventional slide. The chamber or an objective lens may be damaged if the user is not careful. One entire grid on standard hemocytometers with Neubauer rulings can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed.
Cell suspensions should be dilute enough so that the cells do not overlap each other on the grid, and should be uniformly distributed. To perform the count, determine the magnification needed to recognize the desired cell type. Now systematically count the cells in selected squares so that the total count is 100 cells or so (number of cells needed for a statistically significant count). For large cells this may mean counting the four large corner squares and the middle one. For a dense suspension of small cells you may wish to count the cells in the four 1/25 sq. mm corners plus the middle square in the central square. Always decide on a specific counting patter to avoid bias. For cells that overlap a ruling, count a cell as "in" if it overlaps the top or right ruling, and "out" if it overlaps the bottom or left ruling.
Here is how to determine a cell count using a standard hemocytometer. To get the final count in cells/
ml, first divide the total count by 0.1 (chamber depth) then divide the result by the total surface area counted. For example suppose you counted 125 cells (total) in the four large corner squares plus the middle combined. Divide 125 by 0.1, then divide the result by 5 mm-squared, which is the total area counted (each large square is 1 mm-squared). You should get 125/ 0.1 = 1250. 1250/5 = 250 cells/mm-cubed. There are 1000 mm-cubed per ml, so you calculate 250,000 cells/ml. Sometimes you will need to dilute a cell suspension to get the cell density low enough for counting. In that case you will need to multiply your final count by the dilution factor. For example, suppose that for counting we had to dilute a suspension of HEK 293 cells 10 fold. Suppose we obtained a final count of 250,000 cells/ml as above. Then the count in the original (undiluted) suspension is 10 x 250,000 which is 2,500,000 cells/ml.
For our experiments we are interested in seeding cells at a concentration of 5 x 105
cells/well of a migration chamber. Given the concentration of cells in your tube, how many uL of the cell suspension would you need to remove 50,000 cells?
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