Migration assay

Mastyugin, V., McWhinnie, E., Labow, M., and Buxton, F., A quantitative high-throughput endothelial cell migration assay, ]. Biomol. Screen., 9, 712, 2004. 

In addition to above-mentioned cytokines, IRIV were also shown to induce expression and secretion of various chemokines, such as IP-10, MIG, and Rantes (Schumacher R, unpublished). Secretion of chemokines is important for the recruitment of immune cells however, the relevance of this in vitro finding has not been addressed by migration assays or by in vivo studies so far. 

Before performing the migration assay, pretreat the cells with inhibitors or the appropriate control solvent (DMSO). 

Salient features of common 2-D and 3-D invasion and migration assays... 

Yarrow JC, Perlman ZE, Westwood NJ et al (2004) A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods. BMC Biotechnol 4 21... 

Fig. 1. Schematic overview of the tumor spheroid-based migration assay. Tumor spheroids (TS) are transferred from their cuiture vessei or piate into a 96-weii fiat-bottomed migration piate pre-coated with an extraceiiuiar matrix (ECM) protein of choice (in this case, geiatin). Digital images of the spheroids are then captured at t=0 and once every 24 h for a period of up to 72 h, exemplified here by CAL spheroids. Image analysis software is used to calculate the spheroid size and extent of migration. Scale bar=100 p.m.

Tumor Spheroid-Based Migration Assay 1. Appropriate culture medium for the specific tumor cell line(s) under test. 2. 96-well flat-bottomed tissue culture-treated plates. 

For the tumor spheroid-based migration assay, prepare the EC... 

Transfer the plate to a cell culture incubator. Four days later, visually confirm that the EC monolayer is confluent and proceed with the migration assay. 

Import the migration assay images into the image analysis software and select the calibration settings depending on the microscope objective (lOx or 4x) used to obtain the images. 

For the tumor spheroid-based migration assay on EC mono-layer, first aspirate the medium from the EC monolayer 96-well plate and then dispense 200 pL/well of fresh, warm EC culture medium. Then follow the procedure described in Subheading 3.4. 

For highly motile cell lines, the concentration of FBS for the migration assay is kept below that used in standard culture medium (generally 10%). The reasons for this are (1) to slowdown proliferation, thus avoiding effects of proliferation complicating assessment of migration and (2) to reproduce a nutrient deprivation stress which tumor cells may be subjected to in vivo. 

Measurement of leukocyte motility and che-motaxis parameters using a quantitative analysis of the under-agarose migration assay (with D. Lauffenburger). Math. BioscL 44, 121-138 (1978). 

Another critical step is the placement of the filter to the filled wells of the chamber. Caution should be utilized when placing the filter on the filled wells as excessive movement may result in contamination between the wells. Furthermore, in lymphocyte migration assays, matrix-coated filters should be coated at least 2 h prior to performing the assay. The filter should be extensively washed and thoroughly dried prior to the placement of the filter in the chamber. A wet filter may permit leaking between the wells of the chamber. In addition, nonbound extracellular matrix proteins must be removed by extensive washing as residual matrix may result in a loss of adhesion to the filter. 

Finally, several peptide growth factors are well established chemoattractant for endothelial cells, and can be used as positive controls in migration assays. These include bFGF and VEGF in the concentration range 10-100 ng/mL. 

Fig. 1. The chemotaxis of human umbilical vein endothelial cells in response to bFGF (50 ng/mL), VEGF (50ng/mL), and the chemokines IL-8 (50 nM) and RANTES (100 nM). The assay was carried out using filters with a pore size of 8 pM, and the migration assay was carried out for 6 h. The results are given as area of the digitized image of the migrated cells (pixels), using a Sony video camera and a Kontron Vidas image analysis system.

Fig. 11.6. Icmt inhibition impairs activation of RhoA and Racl GTPases and impacts on RhoA- and Racl-mediated cell migration. A, thrombin-mediated activation of RhoA. Cells were treated with thrombin (-f) or vehicle (—) for 15 min, whereupon levels of bound and total RhoA were determined. B, EGF-mediated activation of Racl. Cells were treated with EGF (-f) or vehicle (—) for 15 min, whereupon lysates were prepared and levels of bound and total Racl were determined. C and D, RhoA rescues directed migration. MDA-MB-231 cells were treated for 3 days with cysmethynil Cysmeth) or vehicle Cont) and recombinant adenovirus carrying HA-tagged RhoA or GFP was introduced as indicated. Cells were harvested and transwell migration assays were conducted. E and F, Racl rescues random migration. MDA-MB-231 cells were treated as described above, except that recombinant adenovirus carrying HA-tagged Racl or GFP was introduced. This figure is reproduced from Ref. [28].

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