Migrated cells counting

Air-dry the filter, apply immersion oil, and count the stained nnclei of migrated cells. 

Cells that have migrated are counted in at least 10 high powered fields (x400) using a standard microscope, and the mean value calculated. 

Dilute Matrigel in cold distilled water to 50 /xg in 50-100 /xl. Apply 25-50 /xg Matrigel to the filters, dry under hood, and reconstitute with serum-free medium. Place a solution of chemoattractant in the lower compartment of the Boyden chamber (in the absence of chemoattractant, very little cell migration occurs over a 6-h period). Place the coated filters in Boyden chambers, and close the chamber. Add to the upper chamber 2 to 3 x 10 cells in appropriate medium containing 0.1% BSA. Incubate at 37°C in 5% CO2 for 4-6 h. Remove the cells from the upper surface of the filter by wiping with a cotton swab or by passing them on tissue paper. Fix the filter (methanol or ethanol) and stain (haematoxylin-eosin or toluidine blue). Count the cells from various areas of the lower filter surface. Alternatively, migrated cells can be solubilized... 

To obtain a direct determination of migrated cells, 10 pi of fluorescent microspheres (Flow-Count fluorospheres Beckman Coulter, Fullerton, CA) are usually added to the medium before flow cytometry analysis as a standard. 

This will allow us to count the migrated adherent cells that have stuck to the filter. Using a microscope, count the migrated cells in five high-power fields (e.g., 20 x) per filter. As seen in Fig. 2, pores appear as randomly scattered round dots, and cells are bigger and purple-colored. 

Figure 4 A monolayer of EA.hy-926 cells was grown on the upper surface of a cell culture insert, and THPl cells were assayed for their ability to migrate. After 2 h incubation at 37 °C, filters were fixed with methanol and stained with hematoxylin, and migrated cells were counted.

Figure 7. Effects of the P-III fraction (A) and sea urchin lectin (SUL-I) (B) on neutrophil chemotaxis. Neutrophils (2 x 10 cells/ml) were incubated at 37°C for 60 min with or without the P-III fraction or SUL-I, and migrated cells were counted. The chemotactic response to FMLP (10 M) was expressed as 100 %. A, data represent the mean of two experiments of triplicate determinations B, data represent the mean SD of 5 to 9 experiments of triplicate determinations.

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