TRP-1 expression was verified by indirect immunostaining of Ad5.TRP-1-infected human HEK293T cells (multiplicity of infection (MOI) = 1) using supernatant of the TRP-1-specific hybridoma TA99, kindly provided by Alan N. Houghton Sloan Kettering-Institutee, New York, NY), together with a goat anti-mouse-Cy3 conjugate (BD Pharmingen, Heidelberg, Germany).
Thus, the presented method is believed to be quite independent of the concentration of infected cells (multiplicity of infection and total cell concentration) in bioreactors.
To infect cells, 1 μl of purified virus was diluted in 500 μl of RPMI 1640 medium before slowly adding to the cells (multiplicity of infection of ∼10) and incubating for 24 h at 37°C with 5% CO2.
Further, retroviral transduction was followed for both cell lines for two reasons: first, this methodallows to statistically adjust the copy number by using a low, defined ratio of recombinant virus particles to the number of infected cells (multiplicity of infection).
Note that the ratio of input virus to target cells (multiplicity of infection) of this experiment is 10-fold higher than that of the experiment shown in Figure 2.
The CP09 mutant strain and T1 wild-type strain were grown for 48 hours at 37°C and washed three times with PBS, resuspended in DMEM to a concentration of 10 CFU/mL, and used to infect J774 cells (multiplicity of infection [MOI]: 10 bacteria: 1 cell) grown to approximately 95% confluence in 24-well tissue culture plates.
To verify the thorough killing of extracellular bacteria under the conditions chosen for the antibiotic protection assay, varying numbers of S. aureus ATCC 25923 were added to subconfluent HEp-2 cell monolayers (multiplicity of infection, MOI: 0.01-100) allowedowed to internalize for periods between one to four hours.
For the in vitro studies, data was extracted on cell type, multiplicity of infection (number of viral particles per cell), cell viability, culture time, arginine concentration in the medium and lysine concentration in the medium, when available.
Briefly, a bacterial suspension was prepared at 10 colony-forming units (CFU /mL in RPMI-HEPES and incubated for 1 h with the cell monolayers (multiplicity of infection (MOI) 20 1).
