B. burgdorferi carrying the inducible bpuR construct were cultured to mid-exponential phase (approximately 107 bacteria/ml) either with and without inclusion of 0.5mM IPTG. Bacteria were harvested by centrifugation and washed 3 times with PBS. Aliquots of each culture were resuspended in PBS + 20mM methyl viologen and incubated for 2 hours at room temperature (Esteve-Gassent et al., 2009 (link)). Control aliquots of each culture were incubated in PBS alone. Bacteria were then washed in PBS, and viability assessed by bacterial LIVE/DEAD stain (ThermoFisher). Cells were analyzed by flow cytometry using a CytoFLEX LX (Beckman Coulter) with 488 nm excitation. Optical filters were set up such that red fluorescence was measured above 630 nm and green fluorescence was measured at 520 nm. For each aliquot, 100,000 events were analyzed. The live and dead cells were counted, and proportion of dead cells calculated. The proportions were graphed from three independent cultures. Results were compared by unpaired t-test.
Bacterial live dead stain
Manufactured by Thermo Fisher Scientific
The Bacterial LIVE/DEAD stain is a fluorescent dye-based tool for differentiating between live and dead bacterial cells. It is designed to provide a rapid and reliable method for assessing the viability of bacterial populations.
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Lab products found in correlation
2 protocols using bacterial live dead stain
1
Quantifying B. burgdorferi Viability
2
Oxidative Stress Assay for B. burgdorferi
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B. burgdorferi carrying the inducible bpuR construct were cultured to mid‐exponential phase (approximately 107 bacteria/ml) either with or without inclusion of 0.5 mM IPTG. Bacteria were harvested by centrifugation and washed 3 times with PBS. Aliquots of each culture were resuspended in PBS + 20 mM methyl viologen and incubated for 2 h at room temperature (Esteve‐Gassent et al.,2009 ). Control aliquots of each culture were incubated in PBS alone. Bacteria were then washed in PBS, and viability assessed by bacterial LIVE/DEAD stain (ThermoFisher). Cells were analyzed by flow cytometry using a CytoFLEX LX (Beckman Coulter) with 488 nm excitation. Optical filters were set up such that red fluorescence was measured above 630 nm and green fluorescence was measured at 520 nm. For each aliquot, 100,000 events were analyzed. The live and dead cells were counted, and proportion of dead cells calculated. The proportions were graphed from three independent cultures. Results were compared by unpaired t‐test.
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B. burgdorferi carrying the inducible bpuR construct were cultured to mid‐exponential phase (approximately 107 bacteria/ml) either with or without inclusion of 0.5 mM IPTG. Bacteria were harvested by centrifugation and washed 3 times with PBS. Aliquots of each culture were resuspended in PBS + 20 mM methyl viologen and incubated for 2 h at room temperature (Esteve‐Gassent et al.,
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