Urine samples from three healthy donors were pooled and centrifuged shortly after to remove sediments, if present. After centrifugation, the urine was filtered through 0.22 µm pore size filters (Millipore, Stericup®, Billerica, MA, USA). Artificial urine was prepared by adding the following components: 10.0 g urea, 5.2 g NaCl, 2.1 g NaHCO3, 1.4 g Na2SO4, 1.3 g NH4Cl, 1.2 g K2HPO4, 1.0 g KH2PO4, 1.0 g peptone, 0.5 g MgSO4·7H2O, 0.4 g citric acid, 0.37 g CaCl2·2H2O, 0.1 g lactic acid, 70.0 mg creatinine, 10.0 mg FeSO4·7H2O, and 5.0 mg yeast extract to 1 L of deionized water. To enhance pathogen growth: 20 mg lactose, 20 mg saccharose, and 560 mg glucose were also added to the artificial urine solution. The artificial urine was sterilized by using a 0.22 µm pore size filter (Millipore, Stericup®, Billerica, MA, USA). The urine and artificial urine were then stored at 4 °C until use or at −80 °C for longer storage.
Stericup
Manufactured by Merck Group
Sourced in United States, Germany
Stericup is a sterile filtration system designed for the rapid filtration of cell culture media, buffers, and other aqueous solutions. It features a fast-flow membrane for efficient liquid filtration and a built-in receiver flask for easy collection of the filtered product.
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Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Plenti cmv rtta3 hygro, by Addgene (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Superose 6 increase 3.2 300 column, by GE Healthcare (2 mentions)
Polyethylenimine (pei), by Merck Group (2 mentions)
Talon beads, by Takara Bio (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Nupage 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Coomassie blue stain, by Merck Group (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cellstack, by Corning (1 mentions)
Steritop filter unit, by Merck Group (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Pcr3.1 mgm csf vector, by Addgene (1 mentions)
Steritop, by Merck Group (1 mentions)
Amicon ultra filter unit, by Merck Group (1 mentions)
Tskgel supersw3000 column, by Tosoh (1 mentions)
38 protocols using stericup
1
Preparation of Synthetic Urine for Research
2
Cultivation and Supernatant Extraction of F. graminearum
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F. graminearum D187, from the culture collection of our lab, was primarily grown on PDA at 28°C under white fluorescent light. Nutrient broth (NB; Disco) was used at 3 ml per 17×100 mm tube for preparing bacterial cultures on an incubator shaker at 28°C and 200 rpm. Agar (Aobox, Beijing, China) at 15 g/L was added to solidify the liquid media. Bacteria strains were recovered from storage in 10% glycerol at −80°C by briefly warming the vial at room temperature and streaked onto Nutrient Agar (NA) plate. To obtain a large amount of bacterial culture supernatant for cyclic lipopeptides, the bacterium was grown in 2 L conical flasks each containing 0.5 L of NB. The culture was started with a 1% inoculum and incubated at 28°C and 200 rpm for 48 h. After the cells were separated by centrifugation, the supernatant was filter sterilized by using a 500-mL Stericup™ fitted with a 0.22 μM GP Express membrane (Millipore Corp., Bedford, Mass.).
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F. graminearum D187, from the culture collection of our lab, was primarily grown on PDA at 28°C under white fluorescent light. Nutrient broth (NB; Disco) was used at 3 ml per 17×100 mm tube for preparing bacterial cultures on an incubator shaker at 28°C and 200 rpm. Agar (Aobox, Beijing, China) at 15 g/L was added to solidify the liquid media. Bacteria strains were recovered from storage in 10% glycerol at −80°C by briefly warming the vial at room temperature and streaked onto Nutrient Agar (NA) plate. To obtain a large amount of bacterial culture supernatant for cyclic lipopeptides, the bacterium was grown in 2 L conical flasks each containing 0.5 L of NB. The culture was started with a 1% inoculum and incubated at 28°C and 200 rpm for 48 h. After the cells were separated by centrifugation, the supernatant was filter sterilized by using a 500-mL Stericup™ fitted with a 0.22 μM GP Express membrane (Millipore Corp., Bedford, Mass.).
3
Isolation and Purification of Membrane Vesicles
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OMVs from V. cholerae were isolated as described previously (Wai et al., 2003 (link); Bielig et al., 2011 (link); Elluri et al., 2014 (link)). MVs from L. monocytogenes were isolated as described in the earlier studies with some modifications (Wai et al., 2003 (link); Bielig et al., 2011 (link); Thay et al., 2013a (link)). Briefly, culture supernatants of L. monocytogenes were obtained by centrifuging the bacterial cultures at 5,000 × g for 30 min at 4°C. The supernatants were filtered through 0.22 and 0.1-μm pore size vacuum filters (Stericup, Millipore) sequentially. The bacteria-free supernatants were then ultracentrifuged at 125,000 × g for 3 h at 4°C. The pellets were washed (125,000 × g, 3 h, 4°C) and resuspended in 1x PBS, pH 7.4. The pellets were subsequently used as the MV crude preparation or purified further by Optiprep density gradient centrifugation. Purification was performed using 60% Optiprep Density Gradient Medium (SIGMA) as described earlier (Elluri et al., 2014 (link)). All purified fractions were analyzed by TEM as well as by 13.5% SDS-PAGE gel-immunoblotting for LLO.
4
Lentivirus production and HeLa transduction
5
K. pneumoniae Metabolite Enrichment and Purification
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K. pneumoniae MGH 78578 was incubated for 6 h at 37°C in TSB. The culture was then centrifuged at 7,000×g for 10 min and the pellet was washed twice with M63B1 minimum medium. The bacteria were resuspended in M63B1 supplemented with glucose (0.4% w/v). After two washes in this medium, cells were incubated for 24 h at 37°C with orbital shaking. Cultures were centrifuged for 10 min at 7,000×g at 4°C and the supernatants were filtered through a 0.2 µm filter (Stericup, millipore).
The supernatant was concentrated by evaporation with rotary evaporator (Heidolph Laborota 4000 Efficient) coupled to a vacuum pump (ILMVAC LVS 210 T) and extensively dialysed (CelluSep, H1 membrane, 3 kDa cut-off) with gentle agitation at 4°C during 8 days against ultrapure water (3 baths/day).
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K. pneumoniae MGH 78578 was incubated for 6 h at 37°C in TSB. The culture was then centrifuged at 7,000×g for 10 min and the pellet was washed twice with M63B1 minimum medium. The bacteria were resuspended in M63B1 supplemented with glucose (0.4% w/v). After two washes in this medium, cells were incubated for 24 h at 37°C with orbital shaking. Cultures were centrifuged for 10 min at 7,000×g at 4°C and the supernatants were filtered through a 0.2 µm filter (Stericup, millipore).
The supernatant was concentrated by evaporation with rotary evaporator (Heidolph Laborota 4000 Efficient) coupled to a vacuum pump (ILMVAC LVS 210 T) and extensively dialysed (CelluSep, H1 membrane, 3 kDa cut-off) with gentle agitation at 4°C during 8 days against ultrapure water (3 baths/day).
6
Production and Purification of HIV-1 Pseudotyped Viruses
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The HIV-1NL4-3wt plasmid was obtained from Nathaniel Landau (Alexandria Centre for Life Science, NYU, USA), the HIV-1NL4-3ΔEnv plasmid was a kind gift of Oliver T. Fackler (Universitätsklinikum Heidelberg, Germany) and the BlaM-Vpr plasmid was a gift from Thomas J. Hope (Northwestern University, Chicago, USA). HIV-1NL4-3ΔEnv and HIV-1NL4-3ΔEnv (BlaM-Vpr), both VSV-G pseudotyped, or HIV-1NL4-3wt (BlaM-Vpr) stocks were produced by PEI co-transfection of HEK293T cells. Forty-eight hours later, supernatants were collected and filtered through a 0.45 µm Stericup (Millipore). After sucrose cushion (25% in 1x phosphate-buffered saline (PBS_) purification at 24,000 rpm at 4 °C for 1.5 h (Sorvall WX + Ultra series; rotor: SW32, Beckmann Coulter), virus pellets were resuspended in PBS and stored at −80 °C until use.
7
Bacterial Stationary Phase Protein Extraction
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Two and half hours after entry into stationary phase, bacterial cells were separated from the culture supernatant by sedimentation (8,000 × g, 20 min, 4°C). Note that although the growth curves of the different QS mutants were similar in overall profile (i.e., they all exited exponential phase growth at around the same time), they each reached different final optical density values. At the time of harvesting, the PAO1 and AZM-treated cultures reached an OD600 of 6.5, whereas the lasI, rhlI and pqsR mutants reached final OD600 values of 2.7, 4.9 and 3.5, respectively. The cell pellets from 500 ml culture were immediately frozen and stored at -80°C. Culture supernatants (400 ml volume) were passed through a 0.2 μm filter (Millipore Stericup) and trichloroacetic acid (TCA) was added to a final concentration of 10% w/v. After 4 hr at 4°C, the precipitated protein was collected by sedimentation (8,000 × g, 30 min, 4°C) and the pellet was washed three times with 80% acetone to remove residual TCA. Each pellet was resuspended in ASB14 lysis buffer (8 M urea, 2% w/v amido-sulfobetaine 14, 5 mM magnesium acetate, 20 mM Tris-HCl, pH 8.5) supplemented with protease inhibitor cocktail set I (Calbiochem, La Jolla, CA, USA). The pH of the protein samples was re-adjusted to pH 8.5 using NaOH and the protein concentration was determined using the Bio-Rad DC protein assay kit.
8
Isolation and Purification of Bacterial Outer Membrane Vesicles
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Cells were harvested by centrifugation (5,000×g, 30 min, 4 °C) and GMMA-containing culture supernatants were filtered through a 0.22 µm pore-size Stericup (Millipore, Billerica, MA, USA). If required, supernatants were concentrated to a volume of approximately 60 mL. GMMA were collected by ultracentrifugation (186,000×g, 2 h, 4 °C). The pellet was washed once with PBS buffer, resuspended in PBS, and passed through a 0.22-µm-filter [4 , 5 (link)].
9
Outer Membrane Vesicle Isolation
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NOMV were prepared as previously described [21 (link)]: bacteria were grown at 37°C, 5% CO2, in 50 mL of medium (containing yeast extract, casaminoacids, and lactic acid), to stationary phase. Cells were harvested by centrifugation at 4,000 g, and the supernatant was filtered through a 0.22 μm pore size Stericup (Millipore, Billerica, MA, USA). NOMV were collected by ultracentrifugation of the filter-sterilized culture supernatant (186,000 g, 2 h, 4°C, using Beckman polystyrene tubes). After ultracentrifugation, the NOMV-containing pellet was resuspended in phosphate buffered saline (PBS), and sterile-filtered through a syringe filter with 0.22 μm pore size. NOMV concentrations were determined based on the total protein content, as measured by Lowry assay, in comparison to a standard curve obtained with bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) [28 (link)]. For protein analysis, NOMV were separated by SDS–PAGE using NuPAGE 12% Bis-Tris Protein Gels, and NuPAGE MOPS SDS Running Buffer (Invitrogen, Carlsbad, CA, USA). Total proteins were stained with Coomassie Blue stain (Sigma-Aldrich). fHbp was detected by Western blot using a polyclonal antibody raised in mice against recombinant fHbp v.1 ID 1. All the NOMV used in this study are listed in detail in Table 2 .
10
Preparation and Treatment of Water-Accommodated Fractions
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