Millicel ez slides

One strain of each pathotype detected was tested for adherence patterns. HeLa cells were seeded on tissue culture plates in Dulbecco’s Modified Eagle Media (DMEM) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) at 37 °C in 5% CO₂ until sub-confluence. Then, 5 mL of FC Wash solution with 0.25% trypsin solution was added, incubated for 3 min at 37 °C, and decanted. Fresh DMEM + 10%FBS was added. Cells were adjusted to 5 × 10⁴/mL and 425 µL were seeded on each well of an eight-well Millicel^®EZ slides (Merck Millipore Ltd., County Cork, Ireland). The slide was then incubated overnight at 37 °C in 5% CO₂. HeLa cell monolayers were washed with sterile PBS. After washing, 88.5 µL of bacterial suspension (50,000,000 UFC/mL) in DMEM were added to each well (MOI 1:20, HeLa: Bacteria) and incubated for two h at 37 °C in 5% CO₂. After incubation, wells were washed twice with PBS. Methanol was used to fix cell monolayers for 10 min and samples were stained with Giemsa. The adhesion patterns were observed [35 (link)].

HeLa cells were seeded on tissue culture plates in Minimum Essential Media (MEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) at 37 °C in 5% CO₂ until sub-confluence. Then, 5 mL of FC Wash solution with 0.25% trypsin solution was added, incubated 3 min at 37 °C and decanted. Fresh MEM + 10%FBS was added. Cells were adjusted to 5 × 10⁴/mL, 425 µL were seeded on each well of an eight-well Millicel^® EZ slides (Merck Millipore). The slide was then incubated overnight at 37 °C in 5% CO₂. HeLa cells monolayers were washed with sterile PBS. After washing, 250 µL of bacterial suspension in MEM supplemented with 1% mannose were added in each well (1:20) and incubated for 2 h at 37 °C in 5% CO₂. After incubation, wells were washed twice with PBS. Methanol was used to fix cells monolayers for 10 min and samples were stained with Giemsa. The adhered bacteria number was directly counted microscopically in at least 14 fields of each well; result is expressed as the average bacteria number per cell [42 (link)].