The isolates were injected into MRS broth and keep warm at 30℃ for 24 hr. The preparation of cell free supernatant (CFS) was by centrifuging the broth at 11,500 rpm for 10 min at 4℃. (Mini Spin, AG 22331; Eppendorf, Hamburg, Germany). Each filtration of isolate supernatant used sterile filter (0.45 µm-pore-size filter; Millipore, Darmstadt, Hesse, Germany) [22 ], and this filtrate was utilized for analysis.
0.45 m pore size filter
Manufactured by Merck Group
Sourced in United States, Germany
The 0.45-µm-pore-size filter is a laboratory equipment used for filtration. It has a pore size of 0.45 micrometers, which allows for the removal of particles, bacteria, and other contaminants from various liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Minispin, by Eppendorf (1 mentions)
Milli q, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Polybrene, by Hanbio Biotechnology (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Hiv 1 p24 antigen elisa 2, by ZeptoMetrix (1 mentions)
Weight exclusion columns, by Merck Group (1 mentions)
Milli q plus system, by Merck Group (1 mentions)
Avastin, by Genentech (1 mentions)
15 protocols using 0.45 m pore size filter
1
Preparation of Cell-Free Supernatant
2
Determination of Free Amino Acids in Cheese
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total free amino acid levels of whole cheeses before and after in vitro digestion were determined by the method of Hayaloglu [18 (link)]. Lyophilized cheese samples (0.5 g) were extracted in 10 mL of MilliQ water and filtered through a 0.45-µm-pore-size filter (Millipore, Billerica, MA, USA). The same quantity of lyophilized digestates (0.5 g) was dissolved in 10 mL of MilliQ water and filtered. A quantity of 100 μL of the filtrate was diluted into 1 mL with H2O, and 2 mL of a Cd-ninhydrin reagent (0.8 g ninhydrin was dissolved in a mixture of 80 mL ethanol and 10 mL glacial acetic acid, followed by the addition of 1 g CdCl2 dissolved in 1 mL of distilled water) was added. The Mixtures were vortexed and heated at 84 °C for 5 min and cooled in ice-water, and the absorbance was determined at 507 nm. The results were expressed as miligram Leu per gram of lypohilized WSF by reference to a standard curve which was first prepared using Leu (Sigma Chemical Co., St Louis, MO, USA) at various concentrations (0.050–0.500 mg Leu mL−1 water).
3
Separation of Cheese Protein Fractions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The water-soluble (WSF) and water-insoluble protein fractions (WINF) of cheese were separated according to the following procedure: a grounded cheese (15 g) was extracted in 45 mL of Ultrapure water (Ultrapure water system, SG ver.1.11, Waters, Milford, MA, USA) tempered at 40 °C. To preserve the extract, a drop of formaldehyde was added. The extraction was carried out in an ultrasound bath (Clifton, UK) for 90 min. After that period, the extract was cooled in the freezer for 1 h and centrifuged for 15 min at 4000× g (Janetzki, Prague, Czech Republic). Then, the upper layer was carefully removed, and the supernatant was filtered through Whatman No 1. To further remove any impurities, the obtained filtrate (WSF) was filtered through a 0.45-µm-pore-size filter (Millipore, Billerica, MA, USA) and lyophilized. The precipitate (WINF) was rinsed out with three portions of 5 mL of Ultrapure water. To remove any residual lipids, the WINF was treated with n-hexane for one hour, filtered through Whatman No.1, dried at room temperature overnight and lyophilized.
4
Glucose Consumption in S. aureus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glucose yield was measured in milligrams of glucose consumed per milligram (dry weight) of biomass for S. aureus COL, our primary laboratory strain. Glucose consumption was monitored by enzymatically (R-Biopharm) determining glucose in 200-µl cultures of CDM plus 25 mM glucose over a 4-h period following NO exposure or during a 4-h period during aerobic or anaerobic growth at mid-exponential phase. Dry-weight biomass was determined by vacuum filtering 100 ml of mid-exponential-phase S. aureus COL culture (OD660 of 1) in triplicate through a 10-cm Millipore 0.45-µm-pore-size filter. The filter was then baked overnight at 65°C. Weights were averaged, and the weights of baked sterile filters were subtracted to yield an average dry weight of an S. aureus cell of ~2.8 × 10−13 g. While S. aureus LAC (used for animal experiments) exhibits similar elevated glucose consumption under nonrespiratory conditions, the dry-weight biomass of this strain per OD unit was not directly determined, but it is not expect to differ significantly from that of COL.
5
Quantitative Detection of Methylotrophs in Drinking Water
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To study the presence of methylotrophs and most probable number (MPN) of them, drinking water samples were cultured in mineral salts medium containing: KH2PO4, 1.36 g; Na2HPO4, 2.13 g; MgSO47H2O, 0.3 g; (NH4) 2SO4, 0.5 g; CaCl22H2O, 1.99 mg; FeSO47H2O, 1.0 mg; MnSO4H2O, 0.35 mg; Na2MoO42H2O, 0.5 mg in 1000 mL distilled water with pH, 7.2 and up to 0.5% V/V (0.12 mol/L) of methanol (Merck, Germany) (9 (link), 10 (link)). As the number of bacteria in the samples of treated drinking water is low, 3 L of water was first filtered by 0.45 µm pore size filter (Millipore, USA). Then, to separate the bacteria from the filter surface, it was transferred into a sterile Erlenmeyer flask and washed in 100 mL sterile distilled water with shaking for 30 minutes. Fifteen tubes of the medium were prepared and each set of 5 tubes inoculated with 10, 1, and 0.1 mL of water, which the filter was washed in. To prevent evaporation of alcohol from the medium, tubes were sealed with parafilm and incubated at 30ºC for 20 days. Later, the tubes with turbidity or pellets (indicative of bacterial growth) were selected for isolation of methylotroph bacteria and subsequent experiments.
6
PRMT5 Gene Knockdown using Lentivirus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A lentivirus vector suppressing PRMT5 gene expression was designed and synthesized by Syngen Tech (Beijing, China). The shRNA-PRMT5 and control shRNA were designed according to the PRMT5 gene sequence. The shRNA-PRMT5 sequence was 5'-CAGGAAGAGGGCCTATTTCCC-3'; the control shRNA sequence without significant homology was 5'-TAATTGTCAAATCAGAGTGCTT-3'. Lentivirus was produced by transfection of the plasmids into 293T cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. After transfection for 48 or 72 h, lentivirus was harvested and passed through a 0.45-µm pore size filter (Millipore).
Next, the virus particles were concentrated and stored at −80 °C. To construct the stable cell line, SW780 was infected by the virus with polybrene (8 µg/mL, HanBio Biotechnology, Pudong, China). At 48 h postinfection, puromycin (1 µg/mL, Sigma–Aldrich, St. Louis, MO, USA) was used to screen the stable cell line.
Next, the virus particles were concentrated and stored at −80 °C. To construct the stable cell line, SW780 was infected by the virus with polybrene (8 µg/mL, HanBio Biotechnology, Pudong, China). At 48 h postinfection, puromycin (1 µg/mL, Sigma–Aldrich, St. Louis, MO, USA) was used to screen the stable cell line.
7
Isolation and Functionalization of Salmonella OMVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of HbpD-SpC-decorated OMVs from Salmonella cells was described previously.21 (link) In short, bacteria carrying the plasmid pEH3-HbpD-SpC were grown at 30°C and induced for expression of HbpD-SpC in the presence of 0.1 mM IPTG. After induction for 16 h, cultures were subjected to centrifugation (6,000 × g, 10 min, 4°C) to remove the cells. The spent medium was passed through a 0.45 µm-pore-size filter (Millipore) and centrifuged (235,000 × g, 60 min, 4°C) to sediment the OMVs. The OMVs were resuspended in PBS containing 15% glycerol.
Purified SpT2-tagged antigens were incubated for 18 h at 4°C in 4-fold molar excess over HbpD-SpC, exposed at the OMV surface. After 5-fold dilution with PBS, the reaction mixtures were passed through a 0.45-µm filter to remove potential aggregates. Antigen-conjugated OMVs and control OMVs not incubated with antigen were sedimented by ultracentrifugation (208,000 × g, 75 min, 4°C) after incubation in high-salt washing buffer (PBS, 550 mM NaCl) to remove the contaminants.
Purified SpT2-tagged antigens were incubated for 18 h at 4°C in 4-fold molar excess over HbpD-SpC, exposed at the OMV surface. After 5-fold dilution with PBS, the reaction mixtures were passed through a 0.45-µm filter to remove potential aggregates. Antigen-conjugated OMVs and control OMVs not incubated with antigen were sedimented by ultracentrifugation (208,000 × g, 75 min, 4°C) after incubation in high-salt washing buffer (PBS, 550 mM NaCl) to remove the contaminants.
8
Prophage-free Strain Generation via Mitomycin C
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 12/111ΔphiA prophage-free strain was obtained from the wildtype (WT) 12/111 strain following exposure to 1 µg/mL mitomycin C for 2 h, using a protocol described by Domelier et al. [20 (link)]. After exposure to mitomycin C, the bacterial pellet was resuspended in 1 mL mTH, diluted to 10−7 and plated on a TSH agar plate. After incubation at 37 °C for 18 h, colonies were screened for prophage loss by PCR [16 (link),18 (link)]. Whole-genome sequencing of the 12/111ΔphiA strain and pairwise comparison with the WT 12/111 strain were performed to check the absence of prophage, the closure of the insertion site where 12/11phiA was initially inserted, and the absence of secondary mutation. We searched for phage production following exposure of WT 12/111 to mitomycin C. For this purpose, after exposure to mitomycin C, the bacterial culture was centrifuged (1500× g; 15 min) and the supernatant filtered using a 0.45 µm-pore-size filter (Millipore, Bedford, MA, USA), treated with DNase (TurboTM DNase, Ambion, Austin, TX, USA) and tested for phage by PCR [16 (link)].
9
Argan Press-Cake Ethanol Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mature Argania spinosa fruits of the spherical type were harvested in July 2012 from the Sidi Ifni region (southwest of Morocco). Voucher samples (MARK10888-1) kept at the regional herbarium of Marrakech (Marrakesh, Morocco) were verified by Prof. Ahmed Ouhammou. The samples were dried at 25 °C, after which the argan fruits were manually peeled, and the nut’s shells cracked. The press-cake was obtained by extracting the oil from argan kernels using the mechanical press Komet DD 85 G press (IBG Monforts Oekotec GmbH & Co. KG, Mönchengladbach, Germany).
APC (10 g) was extracted with 100 mL ethanol 70% for 2 weeks at room temperature, after which it was centrifuged (1000× g, 15 min). The supernatant was then filter-sterilized using a 0.45 µm pore size filter (Millipore, Billerica, MA, USA) and kept at −80 °C in a freezer until use.
APC (10 g) was extracted with 100 mL ethanol 70% for 2 weeks at room temperature, after which it was centrifuged (1000× g, 15 min). The supernatant was then filter-sterilized using a 0.45 µm pore size filter (Millipore, Billerica, MA, USA) and kept at −80 °C in a freezer until use.
10
Fungal Culture Preparation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary culture of the four strains (B76, B252, L2, and L5) was prepared by mixing 100 mL of Adamek liquid medium (ALM) with 5 mL of conidial suspension and shaken for 3 days at 150 rpm. The secondary culture (1.0%) was prepared by mixing 250 mL of ALM with 2.5 mL of the primary culture medium and shaken for 6 days at 150 rpm, 25 • C. The mycelium was removed by centrifugation for 15 min at rpm, 4 • C and then the supernatant was filtered through the 0.45 µm pore-size filter (Millipore Corp) to get the filtrate.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!