HeLa and RPE1 cell lines stably expressing fluorescent proteins were generated using the RIEP lentiviral system. For this, 4 × 106 HEK293T cells were seeded in 10 mL DMEM in a 75 cm2 flask. 1.6 μg pCMV-Eco Envelope (CellBiolabs, RV-112), 3.2 μg pCMVR8.74 and 6.5 μg of the respective lentiviral transfer plasmid encoding the protein of interest were mixed in 1 mL of OptiMEM, mixed with 45 μl PEI (1mg/mL Stock, 25K linear, Polysciences, Inc.) and incubated for 15 min. The transfection mix was then added in a dropwise manner to the HEK293T cells. 36 hr post transfection, the virus-containing supernatant was filtered through a 0.45 μm PES filter (VWR) and transferred onto 6 × 105 HeLa or RPE1 RIEP cells (target cells) in a 25 cm2 flask. HEK293T cells were incubated in fresh DMEM and the infection step was repeated 24 hr later. 48 hr after the first infection, target cells were thoroughly washed with PBS twice and incubated in fresh DMEM containing 6 μg/ml blasticidin for selection of viral integration. Another 48 hr later, the washing step was repeated and cells were FACS sorted for the respective fluorescent marker.
Pes filter
Manufactured by Avantor
Sourced in United States
The PES filter is a membrane-based filtration product designed for use in laboratory environments. It is made of polyethersulfone (PES) material and serves as a high-performance filtration solution. The core function of the PES filter is to remove particulates, bacteria, and other contaminants from liquid samples, ensuring sample purity and integrity. The filter is intended for use in a variety of laboratory applications, but a detailed description of its intended use is not provided.
Automatically generated - may contain errors
Lab products found in correlation
Pcmv eco envelope, by Cell Biolabs (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Pspax, by Addgene (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Toc analyser, by Shimadzu (1 mentions)
8 protocols using pes filter
1
Lentiviral Transduction of Fluorescent Proteins
2
Optimizing Competence and Transformation in S. ferus
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Overnight cultures of wild-type S. ferus were grown in 6 mL of CDM or THY at 37°C with 5% CO2. The next morning, the cultures were centrifuged at 4000×g for 10 min. The culture grown in CDM was sterile filtered with a 25 mm 0.22 μM PES filter (VWR) and transferred to a new test tube. The culture grown in THY was resuspended in 1 mL of prewarmed CDM. The culture was spun again at 13,000×g for 5 min, and resuspended again in 1 mL of CDM. After this, the resuspended culture was diluted to OD600 ~0.05 in the 6 mL of filtered supernatant or 6 mL of fresh prewarmed CDM. Resulting cultures were aliquoted at 500 μL in Eppendorf tubes and incubated for 1 hour at 37°C with 5% CO2 before exposure to the following conditions. For fresh prewarmed CDM cultures: 100 ng of DNA and 1 μM S. ferus XIP, No DNA and 1 μM S. ferus XIP, No DNA and No XIP. For filtered supernatant: 100 ng of DNA and 1 μM S. ferus XIP, No DNA and No XIP. Cultures were incubated for 2 hours after exposure and plated for on THY and THY with required antibiotic for selection. Plates were then incubated at 37°C with 5% CO2 for 2–3 days before colonies were enumerated. TE, TF, and statistical significance were calculated, graphed, and observed as detailed in Transformation of S. ferus and construction of S. ferus strains.
3
Lentivirus Production and Transduction
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All lentiviruses were generated using a second-generation system. Briefly, HEK293T cells were transfected with psPAX2, pMD2G, and the plasmid containing a gene of interest or hairpin using polyethylimine (PEI, Polysciences). psPAX2 was a gift from Didier Trono (Addgene plasmid #12260; http://n2t.net/addgene:12260 ; RRID:Addgene_12260) and pMD2.G was a gift from Didier Trono (Addgene plasmid #12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259).
Viral supernatants were harvested 48 h post-transfection, clarified using a 0.45 μm polyethersulfone (PES) filter (VWR), and frozen at -80°C until use. For transduction, lentiviruses were thawed at 37°C and added to target cells in complete media containing 5 μg/mL polybrene (Sigma) for 24 h. The media was changed to selection media containing 1 μg/mL puromycin or 5 μg/mL blasticidin (Thermo) and cells were selected for 48 h before proceeding with experiments.
Viral supernatants were harvested 48 h post-transfection, clarified using a 0.45 μm polyethersulfone (PES) filter (VWR), and frozen at -80°C until use. For transduction, lentiviruses were thawed at 37°C and added to target cells in complete media containing 5 μg/mL polybrene (Sigma) for 24 h. The media was changed to selection media containing 1 μg/mL puromycin or 5 μg/mL blasticidin (Thermo) and cells were selected for 48 h before proceeding with experiments.
4
Anaerobic Incubation of Canga Sediments
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Preparation of batch incubations was carried out in a Coy anaerobic glove bag (Coy Laboratory Products, Inc., Grass Lake, MI, United States) filled with 3-5% H2, balance N2. Synthetic porewater (SPW) composition was based on characterization of IFC porewaters, and contained 5 mM CaCl2, 0.1 mM Na2SO4, and 0.1 mM KH2PO4. Sodium lactate (5.0 mM) was included as an electron donor and carbon source, and the pH of the SPW was adjusted to either pH 4.75 or pH 6.8 with HCl or NaOH, respectively, which were chosen to approximate the pH of porewaters we have observed in IFCs (Parker et al., 2018 (link)). O2 was removed from the SPW by bringing the liquid to nearly boiling, then cooling under a stream of N2 gas for 45 min. Once cooled, SPW-containing bottles were sealed, transferred to the anaerobic glove bag, and filter-sterilized with a 0.2 μm PES filter (VWR, Radnor, PA, United States) until use in incubation bottles or column experiments. Batch sediment incubations contained 20 g of pulverized canga (equivalent to approximately 120 mmol Fe(III)) and 60 mL of SPW in 160 mL serum bottles that were sealed with butyl rubber stoppers held in place with aluminum crimp seals. Where appropriate, 5 g of sub muros material and associated microbial community was used as inoculum for non-sterile incubations. Incubations were carried out in triplicate.
5
Lentiviral Vector Production in 293T Cells
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The 293T plates were seeded in 100 mm TC dishes and allowed to attach overnight under conditions to reach ~70% confluence after 20 h. The following day, 6.4 μg envelope plasmid (psPAX (Addgene Plasmid #12259)), 1.9 μg pMD2.G (Addgene Plasmid #12260) and 5.6 μg lentiviral expression plasmids of interest—pLKO.1-puro Non-Target shRNA Control Plasmid DNA (Sigma SHC016), shNF1 (TRCN0000039713, TRCN0000039714 (Sigma)—dsRed2-RFP (Addgene 109377), or GFP (Addgene 17448) were combined in deionized water to a total volume of 100 μL in a polystyrene culture tube. Polyethylenimine (41.7 μg, linear MW 25000, Polysciences cat# 23966-1) was added to the plasmid mixture and allowed to incubate for 15 min at room temperature. The plasmid:PEI mixture was added to the 293T plates at 70% confluence in 10 mL fresh complete culture medium and incubated for 18 h at 37 °C/5% CO2. The following day, the medium was aspirated and replaced with 15 mL complete culture growth medium and incubated for 48 h. The medium was removed and filtered using 0.45 μM PES filter (VWR). Lentiviral supernatant was aliquoted for single-use and stored at −80 °C.
6
Lentiviral Construct Transfection in HEK-293T Cells
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HEK-293T cells at 70 to 80% confluence were transfected using 3.3 μg of the target lentiviral construct, 2 μg pSPAX2, and 1 μg pMD2.G with 1 mg/ml polyethylenimine (PEI; Sigma) in serum-free DMEM. After 5 to 6 h, medium was replaced with antibiotic-free DMEM containing 10% fetal bovine serum (FBS) and 2 mM l -glutamine (Gibco). Transfected cells were incubated for 48 h at 37°C to allow lentivirus production. The supernatant media containing viral particles was harvested and filtered through a 0.45-μm polyethersulfone (PES) filter (VWR, Radnor, PA, USA) and aliquoted. Virus was stored at −80°C until use.
7
Anthocyanin-Enriched Grape Skin Fermentation
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The PN skin extract dried leftover (extraction procedure is described in the Sect. 2.3.2) was reconstituted in 80 mL of synthetic grape must (SGM) which was in turn filtered through sterile 0.22 µm PES filter (VWR, Austria). Initial concentration of total anthocyanins in filtered synthetic grape must was determined with HPLC-DAD (Sect. 2.5) and was 93 mg/L (expressed as malvidin-3-glucoside equivalents). Fermentations with S. cerevisiae FPC yeast were carried out in deep well microtiter plates using synthetic grape must with PN skin extract or synthetic grape must with 50 mg/L of malvidin-3-O-glucoside. To the synthetic must solution, a p-coumaric acid was added as well in such amount that its final concentration reached 50 mg/L. This medium (1.35 mL) was distributed into deep well microtiter plates. FPC yeast culture was inoculated into the medium (150 µL). The deep well plate was covered with sterile cover to prevent evaporation. The fermentation spanned for 10 days and was performed in four replicates.
8
Characterizing Snow Composition and Particulate Matter
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For pH, conductivity and chemical properties, the snow (triplicates for each location and snow height) was allowed to slowly melt and equilibrate at room temperature. pH and conductivity were measured with a Multi 340i Multimeter (Cole-Palmer, Weilheim, Germany). For DOC measurements, the melted snow was immediately syringe-filtered through a 0.45-μm PES filter (VWR). Ten mL of the filtrate was acidified with 40 μL of 37 % HCl prior to measurement with a Shimadzu TOC analyser (Shimadzu GmbH, Reinach, Switzerland) .
Soluble anions (NO 3
were measured with a Dionex D-320 Ion Chromatography system and Chromeleon package (Dionex, Sunnyvale, CA), while NH 4
+ was estimated with the colorimetric method of Mulvaney (1996) .
For PM characterization, the glass fibre filters were allowed to dry and stored in boxes at room temperature until analysis. The filters were weighed before and after filtering. The difference in weight expressed the amount of PM contained in the snow, was recorded. The PM weight of each of the filters was corrected according to the original snow sample volume.
Soluble anions (NO 3
were measured with a Dionex D-320 Ion Chromatography system and Chromeleon package (Dionex, Sunnyvale, CA), while NH 4
+ was estimated with the colorimetric method of Mulvaney (1996) .
For PM characterization, the glass fibre filters were allowed to dry and stored in boxes at room temperature until analysis. The filters were weighed before and after filtering. The difference in weight expressed the amount of PM contained in the snow, was recorded. The PM weight of each of the filters was corrected according to the original snow sample volume.
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