In vitro model for choriocarcinoma cell viability, migration and invasion using the Alamar Blue assay S. Al-Nasiry, N. Geusens, C. Luyten, M. Hanssens and R. Pijnenborg* Dept. of Obstetrics & Gynecology, Katholieke Universiteit Leuven, Leuven, Belgium Objectives The current techniques to quantify trophoblast viability, migration and invasion have significant limitations e.g. killing the cells during the assay procedure (MTT test). In this study we applied the technique of Alamar Blue (AB) reduction to quantify in vitro cell viability, migration and invasion through fibronectin-coated filters. BeWo and JEG-3 choriocarcinoma cell lines were used as a model for trophoblast cells. This model was tested in the presence of a reported inhibitor, TGFβ. The main advantages of being high reproducible and non-toxic to cells allow continuous monitoring in culture. This promising assay might prove valuable in for qauantitaive analysis of trophoblast cell viability, migration and invasion. Alamar Blue assay for cell viability/proliferation Method: To determine the optimal seeding density and culture period, cells were cultured in 96-well plate (200µl/well) with 1% FCS at concentrations of 1.5x104 – 1x106 cells/ml in a standard incubator. After 4h, Alamar Blue was added at 10% final concentration. OD’s were read at 540 & 630nm after 1, 2.5, 4, 24, 48, 72, and 168 hours from adding Alamar Blue. Cell viability at each point in time was confirmed by direct microscopy and by performing an MTT test Effect of incubation time and cell density: The % AB reduction increased over culture time, from a starting cell density of 50 x103 cells/ml and higher, and starting from 4 hours after addition of Alamar Blue (Fig.3). Based on these results, we decided for the following experiments to use a cell conc.of 1x105 cells/ml and an incubation time with Alamar Blue of 24 hours. Fig.3 * Effect of TGFβ : After 24h of culture, cells received fresh medium with or without 5ng TGFβ. After another 24 hours of culture, Alamar Blue was added directly into culture media at a final concentration of 10% and OD’s were measured after yet another 24-hours period. TGFβ decreased % AB reduction in cultures of both JEG-3 (67.87 vs 71.71) (p<0.05, n=5) and BeWo cells (55.88 vs 71.43) (p<0.01, n=3) after 24 hour of culture. TGFβ had no effect on % AB reduction in HT-1080 cells. Background Alamar Blue® (BIOSOURCE, Belgium) is an aqueous dye that has been used to assess viability and proliferation of various human and animal cell lines. When added to cell cultures, the oxidized form of the Alamar Blue enters the cytosol and is converted to the reduced form by mitochondrial enzyme activity namely by accepting electrons from NADPH, FADH, FMNH, NADH as well as from the cytochromes. This redox reaction is accompanied by a shift in colour of the culture medium from indigo blue to fluorescent pink, which can be easily measured by colorimetric or fluorometric reading (Fig.1). Alamar Blue assay offers several advantages above commercially available assays, being: Safe to lab personnel: non-radioactive, non-toxic. Does not necessitate killing the cells, cells can be: continuously monitored over time used for further analyses (IHC, flow cytometry, etc) Simple: adding 10% solution to culture media Rapid: reading OD after min 4h incubation. Sensitive and reproducible: from as few as 80 cells. Fig.1 control test +ve control -ve control Alamar Blue assay for cell migration and invasion Method: A 24-well Falcon plate is used as a “feeder tray” in which 12µ Millipore filters with a 12µ-pores are placed. For the invasion assay, the same filters were pre-coated with human fibronectin solution. 400 µl of cell suspension is seeded on top of the filters which were then immersed into the feeder trays (Fig.4). After 4 hours of initial cell attachment, the medium in feeder tray is changed into medium +/- 5 ng of TGFβ. An incubation period of 24 hours is allowed for cells to invade the fibronectin layer and migrate through filter pores Alamar Blue is added to the feeder tray at 10% final concentration and the plate is further incubated at 37°C for 24h. 200µl of conditioned culture medium is transferred into a new 96-well plate. OD’s are read at 540 & 630nm. % MIG (or % INV) = % AB reduction of migrated (or invaded) cells x 100% % AB reduction of cells without filters Test reproducibility: Cell migration and invasion was confirmed by visualizing cells with an inverted microscope on the bottom of the filter after incubation with Alamar Blue and after crystal violet staining. The intra-assay variability for % MIG and % INV was 18% (mean 31%, SD 5.6, n=3) and 6.7% (mean 54%, SD 3.6, n=3), respectively. Effect of seeding density: % MIG increased with increasing cell concentration, 29%, 47% and 59% for 0.5x105, 1x105 and 2x105 cells/ml, respectively, but this slight difference was statistically not significant. Effect of scraping the filters: During the of incubation with Alamar Blue, further migration through filters occurs. To test whether this extended invasion can affect the % AB reduction, we carried out an experiment by which we removed adherent cells by scraping the top of the filters with a sterile cotton swab just before adding Alamar Blue. The “scraped” filters showed lower % INV than the non-scraped filters (6.0 vs 23.9). We concluded that significant invasion still occurs during the 24-hour period in which the cells are incubated with Alamar Blue. Effect of TGFβ: (Fig.5). (Fig.5) (Fig.4) Alamar Blue assay for cell quantification Method: JEG-3 and BeWo cells were brought to a concentration of 1x106 cells/ml and subsequently to serial 1:2 dilutions. The resulting different cell suspensions were cultured into duplicate wells of a 96-well plate (200µl/well), and incubated at 37°C. After an initial 4-hour period allowing for cell attachment, 20µl Alamar Blue solution is directly added to medium resulting in a final concentration of 10%. As negative control Alamar Blue was added to medium without cells. The plate is further incubated for 24 hours at 37°C. The absorbance of test and control wells is read at 540 & 630nm with a standard spectrophotometer. The calculation of the percentage of Alamar Blue reduction (% AB reduction) is as follows according to the manufacturer’s protocol: % AB reduction = (εox λ2) (A λ1) - (ε ox λ1) (A λ2) x 100 (εred λ1) (A’ λ2) - (ε red λ2 ) (A’ λ1) where ε λ1 and ε λ2 are constants representing the molar extinction coefficient of AB at 540 nm and 630 nm respectively in the oxidized (εox) and reduced (ε red) forms. A λ1 and A λ2 represent absorbance of test wells at 540 nm and 630 nm respectively. A’λ1 and A’λ2 represent absorbance of negative control wells at 540 nm and 630 nm respectively. The values of % AB were corrected for background values of negative controls containing medium without cells. Standard curve of % AB reduction vs. logarithm of cell concentrations: This was linear between 16.6 x103 and 500 x103 cells/ml (Fig.2a), with r2 values of 0.73, 0.81 and 0.81 and p values for slope of 0.014, 0.005 and 0.006, for HT-1080, JEG-3 and BeWo cells respectively. The intra-assay and inter-assay coefficient of variation (+/- SD) was 1.88% (1.12) and 2.94% (0.92), respectively. Effect of medium colour: In order to remove differences in medium colour, the experiment was repeated using culture media filtered with sterile activated charcoal to remove phenol red from culture media. Briefly, after adding heat inactivated FCS to the medium,16.5 mg activated charcoal was added per 1 ml medium, the bottle was thoroughly shaken during 30 minutes, centrifuged at 3000 RPM for 10 minutes, and the supernatant was filtered to yield a colourless medium. The standard curves showed better linearity with r2 values of 0.60 and 0.66 and p values for slope of 0.041 and 0.026, for HT-1080 and JEG-3 cells respectively (Fig.2b). The following experiments were all carried out with charcoal-filtered media. Fig.2a Fig.2b TGF BeWo JEG-3 P value Control Mean(SD 33.75 (16.18) 19.27 (3.45) < 0.05 TGF Mean(SD 14.83 (4.02) 15.33 (4.19) >0.05 Conclusions Alamar Blue assay is safe, simple, rapid and non-toxic to cells, allowing continuous monitoring in culture. The assay for both viability/proliferation and migration/invasion has high reproducibility with low intra-assay variability. Culture medium colour might affect AB reduction, and therefore media should be rendered colourless by charcoal treatment. TGF has variable effects on cell viability and migration and invasion according to the cell line studied, BeWo cells were more responsive than JEG-3 cells. Accordingly, this promising assay might prove valuable in the study of trophoblast cell viability, proliferation, migration and invasion. Corresponding author: salwan.al-nasiry@med.kuleuven.ac.be