Outer membrane vesicles (OMVs) were isolated as reported previously [4 (link)] with some modifications. Briefly, 18 h cultures of M. catarrhalis strains were diluted 50-fold in 500 ml of brain-heart infusion (BHI) broth and incubated at 37°C for 16–18 h with orbital shaking (150 rpm). The cultures were centrifuged at 6600 ×g for 15 min at 4°C. The supernatants were collected and passed through a 0.22 μm pore-size filter vacuum pump (Merck, Millipore). The filtrates were concentrated using 50 or 100 kDa Vivaspin centrifugal concentrators (Amicon Ultra, Millipore) at 5000 ×g for 30 min at 4°C. The concentrated supernatants were subsequently pelleted overnight (100,000 ×g, at 4°C) in an ultracentrifuge (Beckman Coulter Optima). The pelleted OMVs were resuspended in 500 μl of sterile PBS buffer (pH 7.4), aliquoted, and stored at −20°C. The sterility of OMV preparations was confirmed on BHI agar. The protein concentrations in OMV preparations were measured using a Qubit fluorometer or Bradford assay (Sigma-Aldrich Inc.), and the quality of OMV preparations was confirmed in 12% SDS-PAGE.
Qubit fluorometer
Manufactured by Merck Group
The Qubit fluorometer is a laboratory instrument used for accurately measuring the concentration of nucleic acids (DNA, RNA) and proteins in a sample. It utilizes fluorescent dyes that specifically bind to the target molecules, allowing for sensitive and precise quantification. The Qubit fluorometer provides reliable results with a simple workflow and small sample volumes.
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Lab products found in correlation
Bradford assay, by Merck Group (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Sybr green, by Merck Group (1 mentions)
Rnaclean xp, by Beckman Coulter (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Nebnext poly a mrna magnetic isolation module kit, by New England Biolabs (1 mentions)
Chondroitin sulfate, by Merck Group (1 mentions)
Fluoview confocal microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Alexa fluor 488 labeled phalloidin, by Thermo Fisher Scientific (1 mentions)
1 9 dimethylmethylene blue chloride, by Merck Group (1 mentions)
3 protocols using qubit fluorometer
1
Isolation and Characterization of M. catarrhalis OMVs
2
RNA-Seq Library Preparation from Drosophila Brains
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RNA was isolated using magnetic beads (RNAClean XP, Beckman Coulter, A63987) from 10 Drosophila larval brains following the protocol described in (24 (link)). RNA concentration was determined with a Qubit fluorometer (Thermo Fisher Scientific), and integrity was assessed on the Agilent 2100 Bioanalyzer.
RNA poly(A) purification was performed from 0.8 to 1.2 μg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module Kit (NEB, E7490). Then, complementary DNA (cDNA) generation, adaptor ligation, and library amplification were done with NEBNext Ultra RNA II Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina Set 1, 2, and 3 (NEB E7770, E7335, E7500, and E7710, respectively) following the manufacturer’s instructions. Library amplification was performed with SYBR Green (Sigma, S9430) to establish the necessary number of cycles to quantify (Qubit fluorometer) and to check size distribution (2100 Bioanalyzer) and sequencing. Libraries were sequenced in 125-nucleotide paired-end lanes of an Illumina HiSeq 2500 system, obtaining between 27 million and 56 million of reads per sample.
RNA poly(A) purification was performed from 0.8 to 1.2 μg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module Kit (NEB, E7490). Then, complementary DNA (cDNA) generation, adaptor ligation, and library amplification were done with NEBNext Ultra RNA II Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina Set 1, 2, and 3 (NEB E7770, E7335, E7500, and E7710, respectively) following the manufacturer’s instructions. Library amplification was performed with SYBR Green (Sigma, S9430) to establish the necessary number of cycles to quantify (Qubit fluorometer) and to check size distribution (2100 Bioanalyzer) and sequencing. Libraries were sequenced in 125-nucleotide paired-end lanes of an Illumina HiSeq 2500 system, obtaining between 27 million and 56 million of reads per sample.
3
Quantification of Cell-Hydrogel Constructs
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The hydrogel-cell constructs were stained in a solution of Alexa Fluor 488-labeled Phalloidin (0.8 U/ml; Molecular Probes) and 4,6-diamidino-2-phenylindole (DAPI; 2.5 µg/ml; Thermo Fisher) to look at actin filaments and nuclei, respectively. The samples were imaged with an Olympus FluoView confocal microscope with 10X objective with a step size of 10 µm and a total depth of 500 µm. ImageJ software was used to analyze and quantify cell spreading and circularity of the encapsulated cells.
Picogreen/QuanIT DNA Quantification: The DNA quantity of 3 hydrogels per condition and per time point was calculated using the Picogreen DNA QuanIT kit (Invitrogen). The hydrogels were first degraded in a Proteinase-K (0.5 mg/ml; Sigma Aldrich) digestion buffer, mixed with the reagents provided in the kit, and subsequently read using the Qubit Fluorometer.
Dimethylmethylene blue (DMMB) GAG Assay: At each time point, the same digested samples used in the DNA assay were used for the DMMB assay. 1,9-dimethylmethylene blue chloride (Sigma) was dissolved in ethanol and then added to a 0.04 M NaCl/glycine solution, pH 3, for a final concentration of 46 µM DMMB. The samples were loaded into a 96 well plate, along with the DMMB dye solution, and the absorbance was read at 570 nm. The GAG concentration was calculated from a standard curve generated using a serial dilution of chondroitin sulfate (Sigma).
Picogreen/QuanIT DNA Quantification: The DNA quantity of 3 hydrogels per condition and per time point was calculated using the Picogreen DNA QuanIT kit (Invitrogen). The hydrogels were first degraded in a Proteinase-K (0.5 mg/ml; Sigma Aldrich) digestion buffer, mixed with the reagents provided in the kit, and subsequently read using the Qubit Fluorometer.
Dimethylmethylene blue (DMMB) GAG Assay: At each time point, the same digested samples used in the DNA assay were used for the DMMB assay. 1,9-dimethylmethylene blue chloride (Sigma) was dissolved in ethanol and then added to a 0.04 M NaCl/glycine solution, pH 3, for a final concentration of 46 µM DMMB. The samples were loaded into a 96 well plate, along with the DMMB dye solution, and the absorbance was read at 570 nm. The GAG concentration was calculated from a standard curve generated using a serial dilution of chondroitin sulfate (Sigma).
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