Syto 9 green fluorescent nucleic acid stain

UPEC strain CFT073 (ATCC # 700928; United States), Commensal strain K12 MG1655 (ATCC # 700926; United States), Luria–bertani broth (Himedia cat # M1245; India), Agar (Himedia cat # GRM666; India), C. elegans Bristol wild type N2 strain , E.coli OP50, THP-1 cells (ATCC # TIB-202; United States), RPMI-1640 (Gibco Cat# 11875119; United States), Hela cells (ATCC # CCL2; United States), DMEM (Cat#L0104, Biowest, Nuaille, France), fetal bovine serum (Gibco Cat# 10270106; United States), T24 cells (ATCC# HTB-4™; United States), McCoy’s 5A modified Media(Gibco Cat# 16600082),3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-MTT (Invitrogen Cat # M6494; United States), cas9 Protein (Invitrogen Cat # A36497; United States), 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) (Himedia Cat# RM1817, India), N-hydroxysulfosuccinimide (Sulfo-NHS) (Himedia Cat# RM1120, India), HEPES buffer solution (Sigma-Aldrich Cat # 83264; United States), GeneArt™ Precision gRNA Synthesis Kit (Invitrogen Cat # A29377, United States), TRI Reagent (Sigma Cat # T9424; United States), RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Cat # K1622; United States), SYBR Green Master Mix (Appliedbiosystems Cat# A25742; United States), Crystal violet ( SRL Cat# 28376, India) and SYTO9 green fluorescent nucleic acid stain (Invitrogen Cat # S34854, United States) were used.

Real-time fluorescent PCR was used to select the best primer from seven sets on the Roche LightCycler® 480 II system (Germany). The reaction volume is of 20 μL including 1 μL RNA template (48 ng/μL), 1 μL PNRSV-CPR (20 μM), 0.6 μL PNRSV-MBF (20 μM), 0.6 μL PNRSV-DF (20 μM), 0.2 μL PNRSV-BF (20 μM), PNRSV-BR (20 μM), 0.8 μL dNTP (10 mM), 0.6 μL MgSO₄ (100 mM), 1 μL Bst DNA polymerase (8 U/μL), 1 μL AMV reverse transcription polymerase (10 U/μL), 0.5 μL Betaine (5 mol/L), 2 μL 10× Thermopol buffer, 0.5 μL SYTO® 9 Green Fluorescent Nucleic Acid Stain (100 μM) (Invitrogen). Reaction was carried out at a isothermal temperature of 60 °C for 90 min.

The biofilms were visualized by confocal laser scanning microscopy (CLSM) using Syto 9 green fluorescent nucleic acid stain (Invitrogen, CA, USA) as previously described (12 (link)). The biofilms were cultured on coverslips (22 × 22 mm; Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) in 6-well-plates for 24 h at 37°C. Then, the supernatant was discarded and washed twice to remove planktonic cells. After staining with Syto 9, the biofilms were examined under the microscope (SP8 X, Leica, Germany) with excitation at 488 nm and emission at 495–547 nm.

Pg, A. actinomycetemcomitans strains and Ss were initially incubated separately for 48 hours, 24 hours and overnight respectively, in TSB medium under anaerobic conditions to early stationary phase. The resultant growth was then resuspended in fresh CDM, followed by 10% inoculation into CDM medium and incubation under micro-aerobic conditions for biofilm formation. CDM was prepared as previously described (10.0 mM of NaH₂PO₄, 10.0 mM of KCl, 2.0 mM of citric acid, 1.25 mM of MgCl₂, 100 µM of FeCl₃, 20.0 µM of CaCl₂, 0.1 µM of Na₂MoO₄, 25.0 µM of ZnC1₂, 50.0 µM of MnC1_2, 5.0 µM of CuCl_2, 10.0 µM of CoCl₂, 5.0 µM of H₂BO₃, 1% (w/v) Tryptone, 7.67 µM of Hemin and 2.91 µM of Menadione)^{44 (link)}. Biofilms were incubated in 4-chambered glass coverslip wells (Chambered Coverglass, Nunc™ Lab-Tek™) for 4 days at 37 °C. Cultures were grown anaerobically (0% O₂, 10% CO₂, 10% H₂ and 80% N₂) or micro-aerobically (6% O₂, 7.2% CO₂, 7.2% H₂ and 79.6% N₂) in jars using the Anoxomat^® system (Spiral Biotech, Norwood, MA). Single-species biofilms were stained using SYTO 9 (SYTO™ 9 Green Fluorescent Nucleic Acid Stain, Invitrogen™ Molecular Probes™) and FISH was used to analyze and characterize the composition in multi-species biofilms.

The solid-phase binding of the bacteria to the synthetic peptide was conducted as described previously [21 (link),22 (link)]. In brief, the synthetic peptide (40 μg/mL) was incubated in microtiter plates overnight at 4 °C after dilution in coating buffer (100 mM sodium carbonate, pH 9.6) and then washed twice with the relevant buffers. The samples were added to the G. parasuis suspension (100 μL/well, 5 × 10⁸ bacteria/mL), co-incubated for 2 h at 37 °C and washed twice. To label the G. parasuis adhering to the synthetic peptide, SYTO™ 9 Green Fluorescent Nucleic Acid Stain (Invitrogen, USA) was added at 100 μL/well and incubated for half an hour in the dark at 37 °C and then washed three times and measured in a SpectraMax M2 microplate reader (Molecular Devices, San Jose, CA, USA) at 485 nm absorption and 535 nm emission. The solid-phase adhesion assay was independently repeated 3 times.

SYTO-LAMP reaction was performed by using the P. knowlesi primers (Table 1) that have been reported previously [29 (link)]. A working stock of 50 µM SYTO-9 green fluorescent nucleic acid stain (Invitrogen Corporation, Waltham, MA, USA) was prepared by using distilled water. The 25 μL reaction mixture consisted of 4 µL DNA template, 8.45 µL distilled water, 2.5 µL of 10× isothermal amplification buffer, 1.5 µL of magnesium sulfate (MgSO₄), 3.5 µL of deoxynucleoside triphosphate (dNTPs) (10 mM), 1 µL of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, Ipswich, MA, USA), 40 pmol of FIP and BIP each, 10 pmol of FLP and BLP each, 5 pmol of F3 and B3 each, and 0.5 µM of SYTO-9 green fluorescent nucleic acid stain. DNA amplification was carried out at 65 °C in BioRad CFX96 real-time PCR machine (BioRad Laboratories, Hercules, CA, USA) for ~60 min.
In order to obtain the most suitable concentration of SYTO 9, different concentrations (0.25, 0.5, 1, and 1.5 µM) were tested. The SYTO 9 was diluted from 5 M stock to the desired final dilutions with sterile distilled water. The SYTO-LAMP assays with different concentrations were performed in duplicate and repeated twice.

Caffeine was purchased from Sigma-Aldrich (cat # C0750, Saint Louis, MO, United States). The bacterial strains used were CFT073, UPEC strain (ATCC # 700928; United States) and K12 MG1655 (ATCC # 700926; United States) which were first streaked onto fresh plates from glycerol stocks stored in −80 °C. Single colony was then picked and grown in Luria–Bertani (LB) (SRL Cat# 29817, India) media at 37 °C for 24 h with continuous shaking at 150 rpm to obtain primary culture. For all the experiments 10⁷ CFU/ml E.coli suspensions were used to ensure similar number of bacteria in each experiment. Other chemicals used were 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-MTT(Invitrogen Cat # sM6494; United States), Trypsin–EDTA solution (1×) (Himedia Cat # TCL048)for cell viability of bacteria. For RT PCR, TRI Reagent (Sigma Cat # T9424; United States) was used, Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific Cat # K1622; United States) and SYBR Green Master Mix (Applied biosystems Cat# A25742; United States) were used. Crystal violet (SRL Cat# 28376, India) was used for CV assay and SYTO9 green fluorescent nucleic acid stain (Invitrogen Cat # S34854, United States) for fluorescence microscopy.