Experiments were performed in accordance with the Animals Scientific Procedures Act (1986) and were approved by the local Ethical Review Committee. Barrier-bred female 8–12 week old Balb/c mice (Charles River UK Ltd) were anaesthetised by intraperitoneal injection of 100 mg kg−1 body weight ketamine (Ketaset; Fort Dodge Animal Health, Southampton, UK) and 10 mg kg−1 body weight xylazine (Rompun; Bayer, Newbury, Berkshire, UK) and inoculated with M. smegmatis or M. tuberculosis by endotracheal aerosol application of a total volume of 25 µl using a Microsprayer® (PennCentury, Philadelphia, PA, USA) as previously described [74] (link).
Assessment of bioluminescence (photons s−1cm−2 steridian [sr]−1) from living animals was performed using an IVIS® Spectrum system (Caliper Life Sciences, Alameda, USA) which consists of a cooled charge-coupled device camera mounted on a light-tight specimen chamber. Prior to bioluminescent imaging, mice were anaesthetised with 4% isoflurane. Luciferin dissolved in sterile D-PBS was then administered to animals inoculated with FFluc expressing strains [20 µl of 15 mg ml−1 (47 µM) or 30 mg ml−1 (94 µM) luciferin via the intranasal route, or 300 mg kg−1 or 500 mg kg−1 body weight by intraperitoneal injection]. To image mice infected with Gluc expressing M. smegmatis 50 µl of 0.48 mM or 0.96 mM coelenterazine (prepared by diluting the 10 mM stock in sterile D-PBS just before use) was intranasally administered (10 or 20 µg per mouse respectively), or 150 µl of 0.16 mM coelenterazine via the intraperitoneal route (10 µg). Mice were placed into the camera chamber of the IVIS® Spectrum imaging system where a controlled flow of 2.5% isoflurane in air was administered through a nose cone via the IXG8 gas anaesthesia system (Caliper Life Sciences). A grayscale reference image was taken under low illumination prior to quantification of emitted photons over 30 s to 5 min, depending on signal intensity, using the software program Living Image (Caliper Life Sciences) as an overlay on Igor (Wavemetrics, Seattle, WA). For anatomical localisation, a pseudocolour image representing light intensity (blue, least intense to red, most intense) was generated using the Living Image software and superimposed over the grayscale reference image. Bioluminescence within specific regions of individual mice was also quantified using the region of interest (ROI) tool in the Living Image software program (given as photons s−1). Animals were imaged immediately after inoculation, to assess the success of the delivery, and 24 h post-infection. Animals inoculated with ffluc- or gluc- expressing M. smegmatis were imaged at different time points after substrate administration for up to 3 h.
Assessment of bioluminescence (photons s−1cm−2 steridian [sr]−1) from living animals was performed using an IVIS® Spectrum system (Caliper Life Sciences, Alameda, USA) which consists of a cooled charge-coupled device camera mounted on a light-tight specimen chamber. Prior to bioluminescent imaging, mice were anaesthetised with 4% isoflurane. Luciferin dissolved in sterile D-PBS was then administered to animals inoculated with FFluc expressing strains [20 µl of 15 mg ml−1 (47 µM) or 30 mg ml−1 (94 µM) luciferin via the intranasal route, or 300 mg kg−1 or 500 mg kg−1 body weight by intraperitoneal injection]. To image mice infected with Gluc expressing M. smegmatis 50 µl of 0.48 mM or 0.96 mM coelenterazine (prepared by diluting the 10 mM stock in sterile D-PBS just before use) was intranasally administered (10 or 20 µg per mouse respectively), or 150 µl of 0.16 mM coelenterazine via the intraperitoneal route (10 µg). Mice were placed into the camera chamber of the IVIS® Spectrum imaging system where a controlled flow of 2.5% isoflurane in air was administered through a nose cone via the IXG8 gas anaesthesia system (Caliper Life Sciences). A grayscale reference image was taken under low illumination prior to quantification of emitted photons over 30 s to 5 min, depending on signal intensity, using the software program Living Image (Caliper Life Sciences) as an overlay on Igor (Wavemetrics, Seattle, WA). For anatomical localisation, a pseudocolour image representing light intensity (blue, least intense to red, most intense) was generated using the Living Image software and superimposed over the grayscale reference image. Bioluminescence within specific regions of individual mice was also quantified using the region of interest (ROI) tool in the Living Image software program (given as photons s−1). Animals were imaged immediately after inoculation, to assess the success of the delivery, and 24 h post-infection. Animals inoculated with ffluc- or gluc- expressing M. smegmatis were imaged at different time points after substrate administration for up to 3 h.
Free full text: Click here
