Project description:For determination of reactivity with rDer p 21 and Der p 21 in extracts, a serially diluted rDer p 21 from 100 to 0.00305 µg/mL and 6 different HDM extracts diluted 16 times from their stock concentrations were printed. Human serum albumin, rMBP, HRP and PBS were printed as negative controls. Each allergen and control were printed as a single droplet (~450 pL/drop) in three replicates onto 2D-Epoxy glass slides (PolyAn GmbH, Germany) using sciFLEXARRAYER SX microarray printer (SCIENION GmbH, Germany). The slides were blocked with PBS-T containing 2% BSA (blocking buffer) for 30 min at RT. After that, the slides were incubated for 2 h with MAbs 0.2 µg/mL, diluted with blocking buffer, 80 µL/well). Then the slides were incubated for 30 min with the goat anti-mouse IgG Fc Alexa Fluor® 647 (SouthernBiotech, USA) (1 μg/mL, diluted with blocking buffer, 80 μL/well). The slides were scanned using InnoScan 710 AL microarray scanner (Innopsys, France). The images were analyzed with MAPIX software (Innopsys, France). The average mean ± standart deviation value of each allergen and control was calculated using spots’ median fluorescence intensity (MFI) values
Project description:PaOMVs in the culture supernatant of P. aeruginosa were harvested by ultracentrifugation at 100,000 × g, 4℃ for 90 min using a Himac CP80WX Preparative Ultracentrifuge (HITACHI, Tokyo, Japan). After removal of the supernatant, the pellet was washed twice and suspended in ice-cold sterile phosphate-buffered saline (PBS). PaOMVs were then isolated from other damaged membranes, aggregated proteins and non-membranous proteins by step-gradient ultracentrifugation using OptiPrep™ Density Medium (Sigma Aldrich, St. Louis, MO). After centrifugation at 100,000 × g, 4℃ for 16 h, the substances in all six fractions (F1-F6) were collected, diluted in PBS, and harvested by ultracentrifugation at 100,000 × g, 4℃ for 3 h. The pellet from each fraction was washed twice and resuspended in a suitable volume of ice-cold sterile PBS.
Project description:PBMCs from patients were isolated using 50 mL Leucosep™ tubes (Greiner Bio-One International, Germany) and Ficoll-Paque™ PLUS (GE Healthcare, Sweden). Whole blood drawn into sodium heparin blood collection tubes were diluted 3x with phosphate-buffered saline (PBS) without calcium or magnesium (Lonza, Walkersville, MD). Diluted cell suspensions were carefully layered on Leucosep tubes and centrifuged for 15 minutes at 800 x g at room temperature (RT). Interphase containing PBMCs were harvested and washed with PBS and subsequently centrifuged for 10 minutes at 250 x g at RT before further processing.
Project description:After isolation, islets were cultured in a serum-exosomes-free culture media for one week. Collected culture media were centrifuged first at 300g for 20 min to pellets cells and then at 10000g for 20 min to discard dead cells and cell debris. Exosomes were then isolated from the supernatant by ultracentrifugation at 110000g for 70 min. Exosomes were collected in a minimal volume of PBS,and added three times the volume of Trizol LS to extract exosomes RNA.
Project description:Bone marrow derived cell were isolated from C57BL/6 mice tibia and femur using an aseptic technique. The bone marrow was flushed out of the bone marrow cavity by using a 26-gauge needle with 10ml PBS. Subsequently cell suspension was passed through a 70µm nylon mesh. After a 10 min centrifugation step at 500 x g, cells were incubated in 2ml red cell lysis buffer for red blood cell removal. After 4 min incubation time, lysis was stopped by adding 23 ml PBS. Cells were resuspended in PBS and FACS sorted for CD45 cells using an AriaIII-sorter and 7-AAD as indicator for dead cells. The cell suspensions were counted with Moxi cell counter and diluted according to manufacturer’s protocol to obtain 10.000 single cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10xGenomics).
Project description:Enrolled 24 participants, including twelve renal biopsy-proven T2DN and twelve T2DM, and isolated uEV s by ultracentrifugation to perform adequate parallel microarrays for mRNAs, lncRNAs and circRNAs and sequencing for miRNAs.One hundred mL of first-morning urine was collected.All urine samples were retained and immediately transferred to the laboratory and centrifuged at 2500 x g at 4°C for 15 min using a horizontal rotor (Dynamica V14R Pro, UK) and at 17,000 x g at 4°C for 40 min using a fixed angle rotor (FA15C rotor, Dynamica, UK). Supernatants were filtered using 0.22µm PES filters (Jet Bio-Filtration, Guangzhou, China) and transferred to new centrifuge tubes and stored at -80 degrees. After overnight melting through a 4 degree, ultracentrifuged at 200,000 xg at 4°C for 2h in a fixed angle P45AT rotor (k factor= 130, CP100NX, Hitachi, Tokyo, Japan) and repeat again after resuspending the pellet in PBS. Supernatants were discarded and the pellets were washed in PBS to obtain the uEVs.
Project description:Acinetobacter baumannii were cultured in Brain Heart Infusion medium (BD Bioscience). For normoxic condition, the cultures were shaken at 125 rpm for 24 h, whereas for hypoxic condition, the cultures were placed under static condition for 48 h. Outer membrane vesicles from A. baumannii (AbOMVs) in the culture supernatant from normoxic and hypoxic conditions (referred to nAbOMV and hAbOMVs, respectively) were harvested by ultracentrifugation at 100,000 × g, 4℃ for 90 min using a Himac CP80WX Preparative Ultracentrifuge (HITACHI, Tokyo, Japan). After removal of the supernatant, the pellet was washed twice and suspended in ice-cold sterile phosphate-buffered saline (PBS). AbOMVs were then isolated from other damaged membranes, aggregated proteins and non-membranous proteins by step-gradient ultracentrifugation using 15 to 45% OptiPrep™ Density Medium (Sigma Aldrich, St. Louis, MO). After centrifugation at 100,000 × g, 4℃ for 16 h, the substances in all six fractions (F1-F6) were collected, diluted in PBS, and harvested by ultracentrifugation at 100,000 × g, 4℃ for 3 h. The pellet from each fraction was washed twice and resuspended in a suitable volume of ice-cold sterile PBS.
Project description:Staphylococcus aureus strain ATCC 1718 was inoculated at 0.5% in 400 mL of Brain Heart Infusion broth for 8 h under aerobic conditions. The supernatant was collected by centrifugation twice at 5,000 x g, 4°C for 20 min, filtrated through a 0.45 µm filter to remove all remaining bacterial cells. S. aureus-derived extracellular vesicles (SaEVs) in culture supernatant were precipitated by ultracentrifugation at 100,000 x g, 4°C for 90 min using a Himac CP80WX Preparative Ultracentrifuge (HITACHI, Tokyo, Japan). After washing with ice-cold PBS, the pellet was suspended and applied to discontinuous iodixanol gradient ultracentrifugation. Layers of 40%, 20%, 10%, and 5% iodixanol in 0.25 M sucrose/10 mM Tris, pH 7.5 were prepared using OptiPrep™ [60% (w/v) iodixanol; Sigma Aldrich, St. Louis, MO]. After centrifugation at 100,000 x g, 4°C for 16 h, the substances in all six fractions (Fr1-Fr6) were collected, diluted in PBS, and precipitated by ultracentrifugation at 100,000 × g, 4°C for 2 h. The pellets were then washed and resuspended in an appropriate volume of ice-cold PBS.
Project description:Overnight cultures of S. aureus were diluted to OD600 = 1 and 1 ml of culture was resuspended in 500 μl of either PBS, human plasma, or human serum, and incubated rotating at 37 °C for 30 m. Suspensions were washed 3x with PBS supplemented with 500 40% sucrose and 20 mM Sodium Azide. Cells were incubated with immobilized trypsin (ThermoFisher 20230), suspended in PBS supplemented with 500 40% sucrose and 20 mM Sodium Azide, at 37 °C for 2 h. cells were pelleted, and the supernatant containing protein fragments was analyzed by mass spectrometry to determine proteins.
Project description:Project goal is to identify proteomic profiles of Yersinia ruckeri, the causative agent of enteric redmouth disease in fish. Four strains (SP-05, CSF007-82, 7959-11 and YRNC-10) of Y. ruckeri were isolated from disease rainbow trout, Oncorhynchus mykiss. Strains, SP-05 and CSF007-82, belong to serotype 1 and biotype 1 (motile and lipase positive), while strains 7959-11 and YRNC-10 belong to serotype 1 and biotype 2 (non-motile and lipase negative) and belong to serotype 1. A single colony of each strain was inoculated into tryptic soy broth (Casein peptone, dipotassium hydrogen phosphate, glucose, sodium chloride, soya peptone, Sigma) and grown at 22 °C with shaking (150 rpm). These starter cultures were then diluted with fresh sterile tryptic soy broth to an optical density (OD 600) of 0.10 ± 0.05. Five hundred microlitres of the diluted starter cultures were inoculated in duplicates, into 25 ml of tryptic soy broth. Cultures were grown overnight at 22 °C with shaking (150 rpm) until the late log phase. Cells were harvested by centrifugation, then washed three times with PBS.