The constructed core plasmid (8 μg) and two envelope plasmids, PSPAX2 (6 μg), and PMD2G (2 μg) were co‐transfected into HEK293T cells in a 6‐well plate according to the manufacturer's instructions of Lipofectamine 2000 (11668‐027, Invitrogen). The supernatant was collected at 48 h after transfection and concentrated by using a Centricon Plus‐70 filter unit (UFC910096, Millipore). Lentivirus with titers 108 TU/mL was used in the experiment. The virus was validated in vivo and in vitro as in previous study.4 In in vitro test, 20 μL lentivirus were added in a 24‐well plate containing 1 × 105 HEK293T cells and DMEM without FBS; after 24 h, the transfection medium was replaced with 500 μL fresh complete medium containing 10% FBS; cells were collected at 48 h after culture and then detected the abundance of the target gene. In in vivo test, daily intrathecal injections of lentivirus or vector (1 μL) were performed for 2 consecutive days in naïve or pain mice and then collected samples day 3 after the first injection to detective the expression of the target gene.
Centricon plus 70 filter unit
Manufactured by Merck Group
Sourced in United States
The Centricon Plus-70 filter unit is a laboratory device used for the concentration and desalting of macromolecules such as proteins, nucleic acids, and other large molecules. It employs a centrifugal force to pass the sample through a semi-permeable membrane, retaining the desired macromolecules while allowing smaller molecules and salts to pass through. The unit is designed to facilitate efficient sample preparation and purification in a variety of research and analytical applications.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (1 mentions)
Exfect transfection reagent, by Vazyme (1 mentions)
Lenti x 293t cells, by Takara Bio (1 mentions)
Ultraculture serum free media, by Lonza (1 mentions)
Spin x centrifuge filter tubes, by Corning (1 mentions)
Transit reagent, by Mirus Bio (1 mentions)
Qiaamp minelute virus spin kit, by Qiagen (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
5 protocols using centricon plus 70 filter unit
1
Lentiviral Vector Production and Validation
2
Lentivirus Production and Transduction
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The constructed core plasmid (16 μg) and two envelope plasmids, PSPAX2 (12 μg) and PMD2G (4.8 μg), were cotransfected into HEK293T cells in a 6-well plate according to the manufacturer's instructions of Lipofectamine 2000 (11668-027, Invitrogen). The supernatant was collected at 48 h after transfection and concentrated by using a Centricon Plus-70 filter unit (UFC910096, Millipore, MA, USA). Lentivirus with titers 108 TU/ml was used in the experiment. The assays of lentivirus in vitro and in vivo infection were performed according to a previous study [11 (link)]. Briefly, 20 μl lentivirus and 1.5 μl polybrene (1.4 μg/μl; H9268, Sigma-Aldrich) were added in a 24-well plate containing 1 × 105 HEK293T cells and DMEM without FBS; after 24 h, the transduction medium was replaced with 500 μl fresh complete medium containing 10% FBS; cells were collected at 48 h after transduction.
3
Lentivirus Production for Gene Delivery
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Lentivirus production was carried out as described previously.27 (link) Briefly, the constructed core plasmid (8 μg) and two envelope plasmids, the PSPAX2 (6 μg) vector and PMD2G (4 μg) vector, were co-transfected into 293T cells in a 10 cm dish containing DMEM without FBS. After transfection for 6 h, complete medium containing 10% FBS was used according to the manufacturer’s instructions provided with the ExFect Transfection Reagent (T101-01/02, Vazyme). The supernatant was collected at 48–72 h after transfection and concentrated using a Centricon Plus-70 filter unit (UFC910096, Millipore, MA, USA). Lentivirus with titer 108 TU/ml was used in the experiments.
4
Production of Replication-Incompetent Lentivirus
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To produce live, replication-incompetent lentivirus, Lenti-X 293 T-cells (Clontech) were transfected with 25 μg of the lentiviral transfer vectors, 25 μg of pCMV-Δ8.9 (packaging plasmid expressing gag and pol), and 5 μg of pMDG-VSVG (vesicular stomatitis virus glycoprotein-pseudotyping envelope) using TransIT Reagent (Mirus Bio LLC), following manufacturer's instructions. One day posttransfection, medium was changed to Ultraculture Serum Free Media (Lonza). The third day posttransfection (or two days after media change), vector supernatants were collected and concentrated by ultrafiltration using Centricon Plus 70 filter units (Millipore) and then filtered through 0.45 μm Spin-X Centrifuge filter tubes (Costar). The vectors were aliquoted and stored at −80°C. The viral titer was calculated by performing serial dilutions of virus on target cells and assaying for fluorescent reporter expression after 3 days.
5
Aqueous Sludge Viral DNA Extraction
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The 0.2-µm filter flow-through from the aqueous treatment samples was collected and stored at 4 °C. Dewatered sludge viral DNA extraction followed the bacterial DNA extraction protocol scaled up to 30 g followed by supernatant filtration through 0.2-µm filter repeated twice. For both sets of samples, 140 mL of filtrate was transferred into Centricon Plus-70 filter units (Millipore Corporation, Billerica, MA). Filtrates were concentrated to ~ 250 µL according to the manufacturer’s instructions with additional modifications described here. Each sample was centrifuged at 3000×g for 30 min at 20 °C in 70 mL increments. The supernatant was discarded after each run. The Centricon Plus-70 filter units were then inverted, and the concentrated viral fraction was transferred to sterile tubes and centrifuged at 800×g for 2 min at 20 °C. The ultrafiltrate was pre-treated with 2U of Turbo DNase and 60 µg of RNAse A (ThermoFisher Scientific, Waltham, MA, USA). Nucleic acid was extracted using the QIAamp MinElute Virus Spin kit (Qiagen Sciences, Maryland, MD) following the manufacturer’s instructions including the Qiagen Protease and omitting the carrier RNA step. Nucleic acid was eluted with 50 µL of AVE elution buffer.
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