2.5. Vero Cell Cytotoxicity Assay

Vero cells (African Green Monkey Kidney cells, ATCC CCL-81), obtained from the ATCC, Manassas, Va., were grown in Dulbecco's modified Eagle's medium with low glucose, DMEM-LG (Invitrogen, Carlsbad, CA) with additional 10% fetal bovine serum.

Five ml of each EC broth-fecal suspension was filtered through a 40μ filter, and the filtrate was centrifuged (1000xg/20 min/4^oC) to collect debris-free supernatant/fecal extracts.

The cytotoxicity assay was conducted as previously described with slight modification [60]. Vero cells were seeded at 10⁵ cells/well in 24-well microtiter plates (Costar, Corning, Ma.) and incubated in the presence of 5% CO₂ at 37°C for 24 h until confluency was reached. Serial dilutions (1 : 2 to 1 : 64) of the fecal extracts were prepared in DMEM-LG, and 100 μl of each dilution was added per well of the microtiter plates. Plates were incubated for 2 days at 37°C with 5% CO₂. The cells were microscopically examined for cytotoxicity each day with a final read on the second day. Cytopathic effects (visualized as detached, rounded cells) were numerically scored 1 through 4 corresponding to <25%, 50%, 75%, and >90% cells affected. Control wells with only media were included on each test plate to verify that the cytopathic effects observed were with the fecal extracts or purified toxins alone. Serial dilutions (1 : 2 to 1 : 256) of purified Shiga toxins (Stx-1 and Stx-2; each at a concentration of 50 ng/100 μl) from the NADC stock (NADC, Ames, IA) were tested on the Vero cells separately to validate the procedure.

The toxin neutralization assay was performed as described previously [61] with slight modification to determine whether the observed CPE was caused by Stx1 and/or Stx2 or other undefined factors. Briefly, microtiter plates with Vero cells and serial dilutions of the fecal extracts were set up as described above. However, in this instance, 100 μl of each fecal extract dilution was mixed with 100 μl polyclonal bovine anti-Stx1 or rabbit anti-Stx2 antisera (NADC stock) and incubated at 37°C/110 rpm followed by overnight incubation at 4°C without shaking. The last dilution at which the fecal extracts produced CPE on Vero cells, in the absence of antisera, was selected for this neutralization assay. A 100 μl sample from each “diluted extract-antisera” mix was added per well of the microtiter plates that were incubated and scored for protection (cells lack CPE) or no protection (cells continue to show CPE). Control wells with only media or fecal extract dilutions were included on each test plate to verify the neutralization effects of the extract-antisera mix, if any. Additionally, purified Shiga toxins (as above), at a dilution of 1 : 256, were mixed with antisera and tested on the Vero cells separately to validate the procedure.