Directional motility assays make use of Boyden chambers or Transwell culture inserts with porous membranes that separate cells seeded in the upper chamber from a chemoattractant supplied in a lower chamber. These assays are often time-consuming and are associated with several limitations due to manual counting and inconsistent results; low signal-to-noise ratio and fluorescence interference; and high cost and the need for specific equipment. Here, we describe a simple, direct, and easy ATP luminescence–based motility assay (ALMA), which can be used for 96-well plate quantification.

Cell migration is essential for normal and pathological processes such as development, tissue repair, immune function, inflammation, and tumorigenesis. Quantitative analysis of cell migration has been mainly undertaken with the so-called Boyden chamber system, formed of an upper compartment into which cells are injected, and which is separated from a lower compartment by a porous membrane with pores of various size depending on the cell type under study. Cells can migrate from the upper compartment through the porous membrane, along a concentration gradient formed by a chemotactic factor present in the lower chamber. Quantification of migrating cells depends on the cell type. Nonadherent cells that have migrated to the lower compartment are collected and counted by optical means or flow cytometry, which requires high cell numbers. Migrated-adherent (MA) cells remain attached to the underside of the porous membrane, and their quantification typically requires colorimetric or fluorometric assays (1,2,3,4). Automated technologies involving optical imaging and specific software is being developed within the industry for screening purposes (5). However, these various assays are often time-consuming, and exhibit limitations due to manual counting and inconsistent results, low signal-to-noise ratio and fluorescence interference, and high cost and the need for specific equipment (5). Most available assays, with the exception of fluorescence labeling assays, are limited to a 6-, 12-, or (at best) 24-well-plate format. Recently, an ATP luminescence–based motility-invasion assay (ALMI) was described that provides a reliable and flexible method to quantify cell motility using a luminescence microplate reader (6). However, the multiple steps and transfer of biological material required in this assay considerably complicate its usefulness and increase the related costs and potential bias. Briefly, in the ALMI assay, the cells remaining inside the chamber of the upper surface of the membrane [nonmigrated (NM) cells] are harvested with a sterile cotton swab, which is than inserted into a well of a 96-well plate containing the ATPLite reagent, and the applicator stick is clipped with pruning shears. To harvest MA cells, the blind well chamber is disassembled and the membrane, devoid of NM cells, is placed in a second well containing the ATPLite reagent. The migrated-nonad-herent (MNA) cells are harvested by resuspending the cells in the lower chamber of the blind well, transferring them to an Eppendorf tube, pelleting the cells in a microcentrifuge, and then aspirating the medium and adding the ATPLite reagent. Here we describe a more simple and direct way to carry out ALMI, which we have adapted to 96-well-plate format and which we call ALMA, for ATP luminescence–based motility assay.

The human breast cancer SKBR3 cell line (ATCC, Manassas, VA, USA) was used to set up the ALMA since this cell line was routinely assayed in the laboratory by means of a colorimetric assay (7). Cells (1 × 10⁵/100 µL) were serum-starved for 24 h (to prepare them for optimal response to the chemotactic factor) and distributed in 6.5-mm diameter Transwell inserts (Cat. no. 3422, Corning, Corning, NY, USA) precoated with 25 µg/mL rat tail collagen I (Cat. no. 11179179001; Roche, Meylan, France) (for 2 h at 37°C) and fitted to 24-well plates (Cat. no. 3526; Costar, Avon, France). EGF receptor (EGFR/HER2)–dependent cell motility was then assayed overnight in response to heregulin β (rh NRG-1β1; Cat. no. 396-HB; R&D Systems, Lille, France) and measured by two methods: one using colorimetry and manual counting and the other using ALMA (Figure 1). For the colorimetric method, NM cells from the upper chamber were scraped from the top side of the porous membrane using a cotton swab, followed by cell fixation in 4% paraformaldehyde and staining in 0.1% crystal violet (Cat. no. C3886; Sigma-Aldrich, Lyon, France) (Figure 1, A and B). Cells were counted manually using a microscope (Olympus IMT2; Olympus, France) (Figure 1C). For the ALMA method, migrated cells on the bottom of the insert were detached by incubation in receiver plates containing medium with 0.5% trypsin-EDTA (Cat. no. 25 300-054; Invitrogen, Cergy-Pontoise, France). Optimal cell detachment was checked by crystal violet staining of the membranes, which showed the absence of remaining migrated cells (not shown). Collected cells were then centrifuged (500× g for 5 min; Model no. 5417R; Eppendorf, Le Pecq, France), resuspended in 100 µL medium, and then transferred to an opaque 96-well plate (Greiner Lumitrac 200; Dutscher, Brumath, France), to which 50 µL CellTiterGlo reagent (Cat. no. G7571; Promega, Charboniére, France) was added. The plate was shaken for 2 min and luminescence was recorded after 10 min of incubation time, using a microplate luminometer (Centro LB960; Berthold, Thoiry, France) (Figure 1, A and D). The compared heregulin-induced cell motility, as determined by colorimetry and ATP luminescence, revealed a similar induction (8.2- versus 8.9-fold, respectively, with a Pearson correlation coefficient of 0.94), indicating that ALMA can provide a convenient alternative method to quantify cell motility as compared with manual counting.