Two independent shRNAs against mouse MLK3 (shRNA1, binding site 2266–2285 and shRNA2, binding site 2383–2402) were designed as 97-bp oligomers containing a 20bp targeting sequence embedded in a shRNAmir stem, amplified and cloned into Xho and EcoRI sites of the miRE lentiviral recipient vector pRRL.SFFV.GFP.miRE.PGK.Puro (SGEP) (Fellmann et al., 2013 (link)). The SGEP plasmid containing Renilla shRNA served as a control. Lentiviral vectors were transfected in 293T cells. Viral supernatants were collected after 24 and 48 hr and passed through a 0.45-μm filter (Sarstedt, Germany). Each fresh viral supernatant was used for primary keratinocyte spinfection (1500 g, 30 min). Primary keratinocyte cultures were harvested 72 hr after first transduction.
0.45 μm filter
Manufactured by Sarstedt
Sourced in Germany
The 0.45 μm filter is a laboratory filtration device used to separate particulates and microorganisms from liquids. It features a 0.45 micron pore size membrane that effectively removes bacteria, yeast, and other small suspended particles from aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Model 5110, by Agilent Technologies (1 mentions)
Lindberg blue, by Thermo Fisher Scientific (1 mentions)
Gateway cloning system, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Pullulan, by Merck Group (1 mentions)
Inulin, by Thermo Fisher Scientific (1 mentions)
Starch, by Merck Group (1 mentions)
D maltose, by Merck Group (1 mentions)
D tagatose, by Merck Group (1 mentions)
Myo inositol, by Merck Group (1 mentions)
D lyxose, by Merck Group (1 mentions)
L arabitol, by Thermo Fisher Scientific (1 mentions)
D saccharose, by Merck Group (1 mentions)
L sorbose, by Merck Group (1 mentions)
17 protocols using 0.45 μm filter
1
Targeted Silencing of Mouse MLK3
2
Prophage Induction in Lactococcus lactis
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Small-scale prophage induction trials were performed in 96-well microtitre plates by (in each well) inoculating 200 μl of GM17 broth with 2% fresh overnight culture of a particular L. lactis strain. Chemical induction was performed in early exponential phase (OD600nm∼0.2) by the addition of MmC at a final concentration of 0 (i.e., without MmC; negative control), 1.3 or 3 μg.ml-1. Incubation was continued at 30°C and bacterial growth was followed for 8 h. A final OD600nm reading was obtained 24 h after the lactococcal strains were inoculated in the 96-well microplate, and growth profiles were then generated. In order to obtain cell lysates, MmC-mediated inductions were performed as described before, but at a large scale. Briefly, this involved the addition of 3 μg.ml-1 of MmC (final concentration) to early exponential phase cultures grown in a 50 ml volume of GM17 broth, followed by overnight incubation at room temperature. Cell debris was removed by centrifugation at 7560 × g for 20 min and lysates were then passaged through a 0.45 μm filter (Sarstedt, Nümbrecht Germany).
3
Spontaneous Phage Mutant Generation
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To generate spontaneous mutants of each of the phages, 1 ml of lysate (approximately 1 × 107 pfu of M188, 1 × 108 pfu of MC293) was added in each case to a sample tube containing 5 ml TSB, 400 μl of 0.185 M CaCl2, 200 μl of overnight culture (approximately 2 × 107 cfu) from the sensitive serotype 4b host strain 473, and 600 μl of overnight culture (approximately 6 × 107 cfu) from the insensitive serotype 4c host strain 33,115. The sample tubes were incubated at 37°C for 3 h, after which an additional 400 μl of overnight culture from the sensitive serotype 4b host was added to each, before returning the tubes to the incubator for a further 3 h at 37°C. The resulting samples were then filter-sterilized through a 0.45 μm filter (Sarstedt), after which the filtrates were plaqued, as previously described, against the insensitive serotype 4c host strain 33,115. Plaques observed on the resulting plates were then purified, propagated, and stocked as described previously (Cavanagh et al., 2013 (link)).
4
Quantifying Major Milk Mineral Contents
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The salts content was determined using the freeze-dried samples. In short, ashes were prepared through overnight sample calcination in a furnace at 550 °C, following the AOAC 923.03 method (Lindberg/Blue, ThermoFisher Scientific, USA). The results obtained in ppm were first converted on a molar basis, and the percentage of precipitated salts in each sample on the corresponding initial element content was then calculated.
The recovered ashes were weighted and rehydrated in 1 mL 25% nitric acid, then transferred in a 50-mL volumetric flask to be diluted in HPLC-grade water. The solution was mixed and passed through a 0.45 μm filter (Sarstedt, Nümbrecht, Germany). The specific contents of the main milk elements (Ca, Mg, K, Na, and P) were analyzed using inductively coupled plasma (ICP) optical emission spectrometry (model 5110, Agilent Technologies, Santa Clara, CA, USA), and the results were reported in mM. The Ca/P molar ratio was also calculated.
The recovered ashes were weighted and rehydrated in 1 mL 25% nitric acid, then transferred in a 50-mL volumetric flask to be diluted in HPLC-grade water. The solution was mixed and passed through a 0.45 μm filter (Sarstedt, Nümbrecht, Germany). The specific contents of the main milk elements (Ca, Mg, K, Na, and P) were analyzed using inductively coupled plasma (ICP) optical emission spectrometry (model 5110, Agilent Technologies, Santa Clara, CA, USA), and the results were reported in mM. The Ca/P molar ratio was also calculated.
5
Retroviral Transduction of HsNFS1 Variants
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HsNFS1-FLAG and HsNFS1.R72K-FLAG cDNA sequences (GenScript, custom order) were cloned into the retroviral pBABE-puromycin vector using the Gateway cloning system (Thermo Fisher Scientific). Phoenix Amphotropic cells were transfected with the pBABE constructs using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Retrovirus containing medium was passed through a 0.45-μm filter (Sarstedt) and added to HEK293T cells after addition of polybrene (4 μg/ml). Stably transduced cells were cultured with DMEM high glucose, GlutaMAX (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), and 1% penicillin/streptomycin (Thermo Fisher Scientific) and maintained under puromycin selection (1.5 μg/ml).
6
Retroviral Transduction of 4T1 Cells
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Retroviral plasmids expressing either GFP, a SWAP70-GFP fusion protein, or a SWAP-70 ABM-GFP fusion protein (Ocana-Morgner et al, 2013 (link)) were transfected into the packaging cell line Plat-E cells using polyethyleneimine (PEI) (PolyScience). 8 μg of DNA was mixed with 20 μg of PEI and 500 μl of DMEM without additives. The mix was vortexed and incubated for 20 min at room temperature. The mix was added dropwise to the Plat-E cells in a 10-cm petri dish containing 9.5 ml of additive-free DMEM. After 6 h, the mix was replaced with 10 ml of complete DMEM. After 48 h, the viral supernatant produced by the Plat-E cells was collected and filtered through a 0.45-μm filter (Sarstedt). The viral supernatant was then used to spin-infect 4T1 cells in the presence of 8 μg/ml of polybrene (Merck/Sigma-Aldrich). The viral supernatant was replaced with fresh complete DMEM the next day. The transduced cells were either be sorted by FACS or selected by antibiotics afterward.
7
Profiling Carbohydrate Fermentation Capabilities
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Initial screening of carbohydrate fermentation was performed using the commercial API50® kit (Biomerieux, Basingstoke, UK) following the manufacturer’s instructions. Additionally, growth measurements in the presence of twelve selected carbohydrates (D-tagatose, L-sorbose, myo-inositol, D-lactose, D-saccharose, D-maltose, D-lyxose, pullulan, starch (all products of SigmaAldrich), amygdaline, inulin, L-arabitol (all products of AlphaAesar, Ward Hill, MA, USA) for each of the strains were performed by monitoring OD600nm using a Synergy HT plate reader (BioTek Instruments, Winsooski, VT, USA). Carbohydrate solutions were prepared by the addition of the carbohydrate of interest (1 % w/v) to the MMRS followed by filter sterilisation (0.45 μm filter, Sarstedt, Wexford, Ireland). 500 μL of supplemented MMRS was inoculated with 1 % (v/v) of a bacterial culture grown in MRS at 30°C. The inoculated samples were grown at 30°C and OD600nm readings were taken after 48 h, by placing 200 μL of a culture in 96 well plate. Each assay was performed in triplicate for each of the strains. Significance of differences in growth was tested by One-way Analysis of Variance (ANOVA), followed by Least Significant Test (LSD), performed in R statistical software (https://www.r-project.org/ ).
8
Production of Protein-Transducing Lentiviral Vectors
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Protein-transducing LNPs devoid of vector RNA were generated by standard calcium phosphate transfection of lentiviral packaging plasmids into HEK293T cells seeded the day before in 10-cm dishes at 4 × 106 cells per dish. The amounts of plasmids used for production of particles in 10-cm dishes were pRSV-Rev: 3 μg, pMD2.G: 3.75 μg, and GagPol-encoding plasmid: 26 μg. Protein-transducing IDLVs carrying vector RNA were generated in a similar manner but with the following amounts of plasmids: pRSV-Rev: 3 μg; pMD2.G: 3.75 μg; GagPol-encoding plasmid: 13 μg; and lentiviral transfer vector: 13 μg. One day after transfection, medium was replaced, and two days after transfection, supernatant was harvested by filtration through a 0.45-μm filter (Sarstedt, Nümbrecht, Germany) and stored in aliquots at −70°C. When necessary, concentrated virus preparations were produced by scaling up the production to 15-cm dishes and ultracentrifugation of viral supernatant through a 4-mL 20% sucrose cushion at 25,000 rpm at 4°C for 2 hr followed by resuspension of the pelleted virus in DPBS−/−. The yield of each vector preparation was determined by p24 ELISA using kits provided either by Zeptometrix (Buffalo, NY) or XpressBio (Thurmont, MD) following manufacturers’ protocols.
9
Conditional Medium Preparation for Mass Spectrometry
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Cells were seeded at approximately 80% confluency in 6cm plates in triplicate overnight: 900,000 (HBEC); 1,000,000 (PC-9); 1,000,000 (H1975); 3,500,000 (HCC4006); 7,000,000 (HCC4011); 2,500,000 (H3255). Plates were rinsed twice with DPBS and media was changed to supplement-free KSFM containing 1% Penicillin-Streptomycin (HBEC) or serum-free RPMI-1640 containing 1% Pencillin-Streptomycin (PC-9, H1975, HCC4006, HCC4011, H3255). Plates containing only media were also prepared, and all plates were incubated for 24 hours at 37°C. Conditioned media was collected, centrifuged at 1000 RPM for 5 minutes, at 4°C, and filtered with a 0.45μM filter (Sarstedt) to remove cell debris. The complete 4mL volume of filtered conditioned media was centrifuged in a Vivaspin Turbo 3kDa ultrafiltration unit at 3220xg, at 8°C until media was concentrated to approximately 150-200μL. Concentrated media was buffer exchanged, where samples were centrifuged twice with 4mL 50mM HEPES buffer, pH 7.0, then once with 1mL HEPES at 3220xg, at 8°C to a final volume of 150-300uL. Samples were stored at -80°C until mass spectrometry sample preparation.
10
Preparation of Bacterial Culture Supernatants
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Bacterial culture supernatants were prepared from Streptococcus pyogenes strains 19165 (ATCC, Manassas, VA, USA), 50362 (clinical isolate from a biopsy), Streptococcus dysgalactiae subspecies equisimilis 12394 (ATCC, Manassas, VA, USA), and 5804 (clinical isolate, septic arthritis) [31 (link)]. Clinical isolates were kindly provided by Parham Sendi and Lucy J. Hathaway (Institute for Infectious Diseases, University of Bern, Bern, Switzerland). Bacteria were grown overnight in brain heart infusion (BHI) (Sigma-Aldrich, Saint-Louis, MO, USA) with 10% fetal bovine serum (FBS) (Seraglob, Schaffhausen, Switzerland) at 37 °C. The culture was diluted 1:100 in BHI-FBS (10%) and incubated at 37 °C until reaching an OD540 of 1. Bacterial cultures were centrifuged at 4 °C for 40 min at 4000 rpm. Culture supernatants were filtered through a 0.45 μm filter (Sarstedt, Nümbrecht, Germany), pH adjusted to 7, aliquoted, and stored at −80 °C until further use.
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