Frozen starter culture was diluted 100-fold in 100 ml warm TSB medium and grown at 37°C until an optical density at 590 nm of 0.25. Radiolabeled erythromycin [N-methyl-14C] (50 μCi/mmol, purchased from Bio Trend, Germany) was added at a final concentration of 0.2 μg/ml (Sutcliffe et al., 1996 (link)). Erythromycin uptake was assessed by taking 5-ml samples from each culture every 10 min for 40 min after the addition of [14C]erythromycin, filtering samples with prewet GF/C glass microfiber filters (Whatman), and washing the filters twice with 5 ml of a 0.9% NaCl and 1 μg/ml erythromycin solution (Sutcliffe et al., 1996 (link)). Filters were dried O.N., dissolved in 10 ml of Insta-Gel Plus liquid (Packard) and the [14C] erythromycin cell-associated was counted with a TRI-CARB 2000 CA Liquid Scintillation Analyzer (Packard).
Tri carb 2000 ca liquid scintillation analyzer
Manufactured by Hewlett-Packard
Sourced in United States
The TRI-CARB 2000 CA Liquid Scintillation Analyzer is a laboratory instrument designed for the detection and quantification of radioactive samples. It utilizes liquid scintillation counting technology to measure the energy of radioactive emissions from a variety of samples.
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2 protocols using tri carb 2000 ca liquid scintillation analyzer
1
Measuring Erythromycin Uptake in Bacteria
2
Quantification of Radioactivity in Biological Samples
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Lymph (100-200 µl), plasma (50 µl) and urine (500 µl-1 ml) were aliquoted into a 6 ml scintillation vial. A minimum of three times volume of scintillation fluid (Ultima Gold, Perkin Elmer, VIC, Australia) was added into the tube and the tube was vortexed then analysed for radioactivity using a liquid scintillation counter (Packard Tri-Carb 2000CA Liquid Scintillation Analyzer, CT, USA).
LN and tissue samples were processed for radioactivity analysis as described previously 29 . Briefly, tissue samples were first homogenised with 5 ml milli-Q water using a GentleMACS™ dissociator (Militenyi Biotec, NSW Australia). LNs (intact) and tissue (100 µg homogenate) were dissolved in 0.7 ml and 2 ml Solvable (Perkin Elmer, VIC, Australia), respectively, and incubated at 60ºC overnight. The next day, samples were cooled to room temperature prior to the addition of 200 µl of 30% w/v hydrogen peroxide in water (Science Supply, VIC, Australia). After the mixture stopped frothing, 2 ml (for LN samples) or 10 ml (for tissue samples) of scintillation fluid was added into the tube.
Samples were incubated in the dark at 4°C for 72 h without agitation before radioactivity analysis using a liquid scintillation counter as above.
LN and tissue samples were processed for radioactivity analysis as described previously 29 . Briefly, tissue samples were first homogenised with 5 ml milli-Q water using a GentleMACS™ dissociator (Militenyi Biotec, NSW Australia). LNs (intact) and tissue (100 µg homogenate) were dissolved in 0.7 ml and 2 ml Solvable (Perkin Elmer, VIC, Australia), respectively, and incubated at 60ºC overnight. The next day, samples were cooled to room temperature prior to the addition of 200 µl of 30% w/v hydrogen peroxide in water (Science Supply, VIC, Australia). After the mixture stopped frothing, 2 ml (for LN samples) or 10 ml (for tissue samples) of scintillation fluid was added into the tube.
Samples were incubated in the dark at 4°C for 72 h without agitation before radioactivity analysis using a liquid scintillation counter as above.
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