Non-Kit Based Migration, Invastion and Chemotaxis: Invasion Assays with Adherent Cells

MultiScreen-MIC Filter Plate

The Millipore family of cell culture inserts and insert plates with 3 µm, 5 µm or 8 µm membrane are disposable devices designed to support cell based assays where cells migrate in response to a chemical gradient.
These cells either traverse a barrier such as an ECM (invasion) or migrate and exhibit tube formation (analogous to capillary formation in vivo) within an ECM matrix (angiogenesis). These inserts and insert plates are available for use with a variety of suspension and adherent cell lines and are particularly recommended for use in cell-based functional assays in the drug discovery field in order to screen protein, antibody, and compound libraries.

The protocol shown below is for the 96-well MultiScreen-MIC plate. The Millicell-24 or individual inserts can be substituted in the protocol below. For a helpful table of scaled volumes please see Recommended Working Volumes in the Microporous Membrane-based Cell Culture chapter of this Handbook.

Millipore also offers a broad range of Migration,Invasion and Chemotaxis ready-to-use kits. For information on these kits please consult the appropriate section within this chapter of the handbook.

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Materials and Reagents

MultiScreen-MIC filter plate — Millipore cat. nos. MAMI C3S 10 (3 µm), MAMI C5S10 (5 µm), MAMI C8S 10 (8 ìm)
MDA-MB-231 (Breast Cancer Cell) — ATCC® cat. no. HTB-26
Hema®-3 stain kit — Fisher cat. no. 22122911
Cotton swabs
ECL Cell Attachment Matrix — Millipore cat. no. 08-110
Calcein AM
Sterile single-well tray — Millipore cat. no. MAMC S01 10
MultiScreen-MIC receiver plate — Millipore cat. no. MAMC S96 10
Zeiss® — Axioplan®-2 microscope with KS300 3.0 software for cell counting and Axiovision-2 software for cell imaging
PBS
Squirt bottle
Milli-Q water

Methods

Note: Standard sterile cell culture lab practices must be followed while performing all of these protocols

A. Cell culture

MDA-MB-231 cells and propagate in RPMI supplemented with 10% FBS, 1X HEPES, 1X NEAA, 1X L-glutamine-penicillin/streptomycin. Passage cells once a week at 100% confluency and incubate at 37°C, 5% CO2.

B. Cell expansion and preparation of adherent cells for invasion assay

Expand MDA-MB-231 cells into T-75 flasks for 3–5 days before experiment such that they are about 80–90% confluent the day prior to setting up the experiment.
Starve the cells overnight at 37°C, 5% CO2 in serum-free medium containing 0.2% BSA (v/v).
Thaw extracellular matrix (ECL) overnight at 4°C as per manufacturer’s recommendations.
On the day of experiment, count the cells and adjust the cell concentration to 106 cells/mL in serum-free medium containing 0.2% BSA. Also make a note of the cell viability. Cell viability of >90% is acceptable for setting up migration experiments.

C. Coating procedure on MultiScreen-MIC filter plates for adherent cell invasion assays

Pre-cool MultiScreen-MIC filter plates on ice for 30 minutes. This step can be performed prior to cell counting. Coat the membrane in the MultiScreen-MIC filter plate with 50 µL of ECM (ECL) diluted to a concentration of 800 µg/mL in cold serum-free medium (final ECM concentration is 40 µg/well).

Notes:
- It is critical to maintain cold conditions per manufacturer’s recommendations during coating. All dilutions of extracellular matrix need to be made using pre-cooled tubes. Gently dispense the extracellular matrix using pre-cooled pipette tips if needed to evenly distribute the matrix over the membrane.
- Take care not to damage the membrane.
Place the coated plates in the incubator at 37°C for 30 minutes. Remove the plates from the incubator and place at room temperature for 1 hour. This step ensures good polymerization of the extracellular matrix.

D. Setting up adherent cell invasion and invasion inhibition assay on ECM-coated MultiScreen-MIC filter plates

Separate out the MultiScreen-MIC filter plate with coated membrane and place into a sterile singlewell tray to protect the membrane prior to setting up the experiment.
Set up the invasion experiment. See “Example Template for Invasion Assay” figure for an example template with chemoattractants.
Add cells to upper wells in the MultiScreen-MIC filter plate (placed in the single-well tray). Ensure that cell suspension is evenly distributed across membrane. Add chemoattractants to lower wells in the MultiScreen-MIC receiver plate. Avoid generating bubbles while adding solutions to apical and basolateral compartments.
Columns 1 and 2 will test unstimulated migration in the absence (basal migration) and presence (basal invasion) of extracellular matrix respectively. Column 4 and columns 5–12 will test stimulated migration to factors in serum containing medium in the absence (chemotaxis) and presence (invasion) of extracellular matrix respectively.
For invasion inhibition experiments, add inhibitors at desired concentration to cells prior to loading in upper wells. Example template with chemoattractants/inhibitors is outlined in the “Example Template for Invasion Inhibition Assay” figure, where columns 9–11 will now test for invasion inhibition.
GENTLY assemble the top and bottom plates together. DO NOT SHAKE OR TILT THE PLATE, as this will disturb the concentration gradient.
Incubate this MultiScreen-MIC plate assembly for 24 hours at 37°C. Do not shake plates during incubation.

E. Staining procedure for adherent cell invasion and invasion inhibition assay plates

Toward the end of the incubation period, set up staining solutions from the Hema-3 stain kit in a new MultiScreen-MIC receiver plate. Dispense 150 µL of the Fixative, Solution 1, and Solution 2 in series in separate plates. Set up two additional plates with Milli-Q water.
After incubation, remove plates without shaking or tilting from the incubator.
Remove unmigrated cells in upper wells with a cotton swab. Use gentle pressure to effectively remove cells from the wells. Take care not to puncture the membrane.
After swabbing all wells, gently rinse the wells with PBS using a squirt bottle.
Stain MultiScreen-MIC filter plates as illustrated in the staining procedure diagram.
Optional: Quickly swab upper wells in filter plate with cotton swabs to remove any residual dye prior to drying plates. This minimizes dye interference when counting.

F. Image analysis of stained invasion or invasion inhibition assay plates

The plates in our experiments were counted using a Zeiss Axioplan-2 microscope equipped with KS300 3.0 automated cell counting software. Eleven percent of the membrane surface area was counted and the cell number was extrapolated to the entire membrane (0.3 cm2 surface area). Plates can also be manually counted using a standard microscope with sufficient magnification to view the cells. Place the filter plate on a slide to avoid damage to membrane. If objectives are above the stage, simply flip the plate so membrane side is facing up towards the objective. Calculate the area of counting field in your microscope and count a sufficient number of fields in the membrane corresponding to 5–10% of total membrane surface area.

Note: For additional calculations on how to interpret invasion assay data, please refer to Millipore application note AN1675EN00. Staining Procedure to Detect Adherent Cells on Membrane Underside

Cell Migration Colorimetric Assay Principle

References

Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, McEwan RN. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987; 47(12): 3239–3245.
Kamath L, Meydani A, Foss F, Kuliopulos A. Signaling from protease-activated receptor-1 inhibits migration and invasion of breast cancer cells. Cancer Res 2001; 61(15): 5933–5940.
Marconcini L, Marchio S, Morbidelli L, Cartocci E, Albini A, Ziche M, Bussolino F, Oliviero S. c-fos-induced growth factor/vascular endothelial growth factor D induces angio-genesis in vivo and in vitro. Proc Natl Acad Sci U S A. 1999;96(17): 9671–9676.

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