Germany) fitted with a Zeiss LSM 510 META Confocal scan head Ima

Germany) fitted with a Zeiss LSM 510 META Confocal scan head. Imaging was carried out using the

458/477/488 nm Argon and 543 nm HeNe laser lines and a 63× C-Apochromat® water immersion lens. Live and dead cells in the stained biofilms were quantified using COMSTAT software [18] with the viability of the biofilm obtained by averaging the number of live cells over the entire z-stack [15]. Biofilm thickness was also measured using light microscopy [15]. Total RNA extraction P. gingivalis W50 biofilm and planktonic samples (40 mL) were immediately added to 0.125 volume of ice-cold Phenol solution (phenol saturated with 0.1 M citrate buffer, pH 4.3, Sigma-Aldrich, Inc. Saint Louis, MO). The mixture was centrifuged and the pellet suspended in 800 μL of ASE lysis buffer (20 mM Na acetate, 0.5% SDS, 1 mM EDTA pH 4.2) and transferred SRT2104 into a 2 mL microcentrifuge tube. An equal SGC-CBP30 solubility dmso volume of ice cold Phenol solution was added and the mixture

was vortexed for 30 s before incubation at 65°C for 5 min. The mixture was then chilled on ice for 3 min after which of 200 μL of chloroform was added and mixed by brief vortexing. The mixture was centrifuged at 16,100 × g and the aqueous phase collected and extracted using a Phenol solution/chloroform (1:1 vol:vol) mix. The RNA in the aqueous phase was precipitated by addition of 700 μL of 4 M LiCl and incubated overnight at -20°C. Samples were then thawed and the total RNAs were pelleted by centrifugation. The pellet was washed with cold 70% ethanol, air dried and suspended in 50 μL of 0.1% diethylpyrocarbonate treated water. The samples were then treated with DNase I (Promega, Madison, WI) and purified using RNeasy Mini columns (Qiagen, Valencia, CA) according to protocols supplied by the manufacturer. The quality of the total RNA was verified by analytical agarose gel electrophoresis and the concentration was determined spectrophotometrically. Microarray analyses Reverse transcription reactions contained

mafosfamide 10 μg of total RNA, 5 μg of random hexamers, the first strand buffer [75 mM KCl, 50 mM Tris-HCl (pH 8.3), 3 mM MgCl2], 0.63 mM each of dATP, dCTP, and dGTP, 0.31 mM dTTP (Invitrogen Life Technologies, Carlsbad, CA) and 0.31 mM aminoallyl dUTP (Ambion, Austin TX), 5 mM DTT, and 800 u of SuperScript III reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42°C for 2 h. The RNA was hydrolysed by incubation with 0.5 M EDTA and 1 M NaOH at 65°C for 15 min and the sample neutralized with 1 M HCl before purification of the cDNA with QIAquick columns (Qiagen). The cDNAs were coupled with monoreactive Cy3 or Cy5 (40 nmol) (Amersham Biosciences, Piscataway, NJ) in the presence of 0.1 M NaHCO3 for 60 min at room temperature. The labeled cDNAs were purified using QIAquick columns (Qiagen), combined and vacuum dried. Samples were then suspended in hybridization buffer containing 50% formamide, 10× SSC (150 mM sodium citrate, pH 7.0 and 1.5 M NaCl), 0.

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