It is essential to remove adherent as well as extracellular bacte

It is essential to remove adherent as well as extracellular bacteria in order to determine the invaded population. For this, gentamicin solution was added to all the wells at a concentration of 25 μg/ml and the plate was incubated

for 1 h at 37°C in 5% CO2 to kill the extracellular bacteria (Note : this concentration was based on the MIC value of gentamycin determined against MRSA 43300 which was 16 μg/ml. In addition, after treatment with 25 μg/ml of gentamycin for 1 hour, the supernatant containing killed bacteria was plated out with complete killing (no colonies on incubation) observed). Finally, the epithelial cells were washed thrice with PBS by centrifugation at 1800 rpm for 10 min at 4°C to remove Tucidinostat research buy non associated bacteria. The cells www.selleckchem.com/products/pnd-1186-vs-4718.html were re-suspended in DMEM and then treated with lysis solution (0.025% trypsin and 1% tween 20 in PBS) for 30 minutes at 37°C in 5% CO2. The cell suspension so obtained was suitably diluted and plated on nutrient agar plates. This bacterial count so obtained represented the number of invaded bacteria (I). The difference between the total number of associated bacteria (T) and the number of invaded bacteria (I) was taken as number of adhered bacteria = (T-I) CFU/ml. Results were expressed as % invasion and % adherence. Cytotoxicity

assay To determine the cytotoxic effect of S. aureus cells on NEC, (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction mafosfamide assay was performed as per the method of Saliba et al. [18]. Washed nasal cells, re-suspended in DMEM were seeded in 12 well plate. After addition of bacteria (bacteria: NEC- 10:1), the plate was incubated for adherence to occur. After 6 h of incubation, gentamicin was

added to the wells to kill the extracellular bacteria. To the washed cells, MTT was added (2 mg/ml in PBS) and incubated for 1 h at 37°C in 5% CO2. Supernatant was discarded and cells were treated with 100 μl of absolute ethanol to dissolve the formazan crystals and absorbance measured at 540 nm. The same procedure was repeated at 24 and 48 hours. Suitable control wells containing only epithelial cells without added bacteria were also processed in the same way at all time points. The learn more percentage cytotoxicity was calculated using the following formula: $$ \%\ \mathrmCytotoxicity = \left[1\hbox-\ \left(\mathrmA_540\mathrmof\ \mathrmtest\ \mathrmwell/\ \mathrmA_540\mathrmof\ \mathrmcontrol\ \mathrmwell\right) \times 100\right] $$ Effect of phage on bacterial adhesion, invasion and cytotoxicity on NEC Washed nasal epithelial cells re-suspended in DMEM were seeded in 12 well plate. Bacterial suspension (corresponding to 1 × 108 CFU/ml) was added to nasal epithelial cells (10:1). Following bacterial addition, phage was added at MOI-1 and 10, and the plate was incubated for 3 h at 37°C in 5% CO2.

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