Triplicate 1 ml samples for microbial cell enumeration using flow cytometry (FCM) were fixed with glutaraldehyde (2% final concentration), snap frozen and stored in liquid nitrogen on-board, prior to -80°C storage post-voyage. Prior to FCM analysis, samples were quick-thawed and divided to enable the separate enumeration of bacteria (200 μl) and autofluorescent picophytoplankton (800 μl). Samples for bacterial enumeration were stained with SYBR Green I [1:10,000] (Invitrogen Molecular Probes, United States), while picophytoplankton samples were analyzed unstained. For both sample types, 1 μm diameter fluorescent microspheres (Invitrogen Molecular Probes) were added as an internal reference (Marie et al., 1997 (link); Gasol and del Giorgio, 2000 (link)). Samples were analyzed using a Becton Dickinson LSR II flow cytometer (BD Biosciences), with bacteria discriminated according to SYBR Green fluorescence and side-scatter, while picophytoplankton populations were discriminated according to orange (phycoerthyrin) fluorescence, red (chlorophyll a) fluorescence and side-scatter (Seymour et al., 2007 (link)). All data were analyzed using Cell-Quest Pro software (BD Biosciences).
Sybr green
Manufactured by BD
SYBR Green is a fluorescent dye used for the detection and quantification of double-stranded DNA (dsDNA) in various applications, such as real-time PCR and DNA gel electrophoresis. The dye binds to the minor groove of dsDNA, and its fluorescence increases significantly upon binding, allowing for the sensitive and specific detection of DNA amplification or presence.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Fluorescent microspheres, by Thermo Fisher Scientific (1 mentions)
Sybr green 1, by Thermo Fisher Scientific (1 mentions)
Cellquest pro software, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Lsrii system, by BD (1 mentions)
Sybr green, by Merck Group (1 mentions)
Bioruptor plus, by Diagenode (1 mentions)
3 protocols using sybr green
1
Microbial Cell Enumeration by Flow Cytometry
2
RT-qPCR Gene Expression Analysis
3
Cell Cycle Analysis by SYBR Green DNA Staining
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To assess cell cycle synchronization efficiency and position along the cell cycle, we followed DNA staining of samples for every time point using flow cytometry. Briefly, EtOH-fixed cells (see above) were washed twice with 50 mM Tris-HCl (pH 8), resuspended in 1 mg/mL RNase A for 40 min at 37°C, washed twice with 50 mM Tris-HCl (pH 8), and resuspended in 2 mg/mL Proteinase K for 1-h incubation at 37°C. Then, cells were washed twice with 50 mM Tris-HCl (pH 8) again, resuspended in SYBR Green (Sigma-Aldrich S9430; 1:1000) and incubated in the dark for 1 h at room temperature. Cells were washed again, resuspended in 50 mM Tris-HCl (pH 8), and sonicated in the Bioruptor plus (Diagenode) for three cycles of 10 sec on and 20 sec off at low intensity. SYBR Green fluorescence was measured using BD LSRII system (BD Biosciences).
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