GDF-15 lentiviral shRNA plasmid was purchased from Origene (catalog# TL710232, pGFP-C-shLenti). Lentiviral particles were prepared following manufacturers protocols (shRNA lentiviral packaging kit catalog# TR30037). Lentiviral particles were concentrated using Origene Lenti Concentrator (catalog# TR30025) and isolated astrocytes were transduced at a MOI sufficient to result in >80% transduction 72 hours post exposure.
Lenti concentrator
Manufactured by OriGene
Sourced in United States
The Lenti Concentrator is a lab equipment product designed for concentrating lentiviral particles. It enables the user to efficiently concentrate lentiviral samples through a simple and straightforward process.
Automatically generated - may contain errors
Lab products found in correlation
Lenti x concentrator, by Takara Bio (2 mentions)
One shot stbl3 chemically competent cells, by Thermo Fisher Scientific (2 mentions)
Turbofectin 8.0 transfection reagent, by OriGene (2 mentions)
Lenti x gostix plus, by Takara Bio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Zymopure plasmid maxiprep kit, by Zymo Research (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pgfp c shlenti, by OriGene (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Hek293t 17 cells, by American Type Culture Collection (1 mentions)
Lipofectamine plus, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Evos fluorescence microscope, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using lenti concentrator
1
GDF-15 Lentiviral Transduction of Astrocytes
2
Generating Stable B2M-KO AM-MSCs
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Human genomic B2M sequences were analyzed and selected using the web tool Benchling (https://benchling.com/ ). B2M-specific CRISPR-Cpf1 expression vector was constructed by cloning the annealed oligomers (5′-agatCCGATATTCCTCAGGTACTC-3′ and 5′-aaaaGAGTACCTGAGGAATATCGG-3′) into a pY108 lentiviral vector (Addgene, plasmid #84739). Infectious lentiviral particles were produced as described previously and were precipitated using Lenti Concentrator (Origene, Rockville, Maryland, US) according to the manufacturer’s protocol [24 (link)]. To produce stable B2M-KO-AM-MSCs, resuspended lentivirus in culture media were added to AM-MSCs and were incubated for 24 h in the culture medium. The cell culture medium was replaced with a fresh medium containing 4 μg/ml puromycin and incubation continued for a further 24 h. AM-MSCs in which B2M was knocked out and that did not express MHC I was selected with a BD FACSAria™ III Cell Sorter. B2M-KO was accessed by an Indel sequencing primer (Table S1 ).
3
Pseudovirus Production and Endocytosis Assay
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Stomatitis Virus (VSV)-G pseudovirus (Hu et al., 2020) . For the production of viral particles, we plated HEK293T/17 cells (American Type Culture Collection, Manassus, Virginia, USA) in poly-L-lysine precoated T75 flasks (3 x 10 6 per flask) and transfected with the plasmid constructs pUMVC (MLVgag-pol), pCMV-VSG-G, and pMKO.1 GFP. We transfected a total of 10 µg of DNA (3 µg of gag-pol and VSV-G plasmids and 4 µg of GFP plasmid), using Lipofectamine 3000 (Thermo Fisher Scientific). We collected cell culture media after 48 and 72 hours, and we concentrated viral particles using a Lenti Concentrator (Origene, Rockville, MD, USA). We determined the viral titer in cultures of HEK293T/17 cells using 96-well plates and serial dilution of virus for transduction.
For the endocytosis inhibition assay, we plated HEK 293T/17 cells in 96-well plates (10,000 cells/well). After 24 hours, we pretreated cells with dynasore or dyngo-4a for 30 minutes, in a dose-range, then added pseudovirus. In parallel, we performed infection with mock pseudovirus, which served as a negative control. After 72 hours, we quantified pseudoviral uptake. Infected cells express Green Fluorescent Protein (GFP). We viewed cells with a microscope equipped with epifluorescence, and counted green (infected) cells. We used Hoechst 33342 to stain the nuclei and we calculated the ratio of infected cells to uninfected cells.
For the endocytosis inhibition assay, we plated HEK 293T/17 cells in 96-well plates (10,000 cells/well). After 24 hours, we pretreated cells with dynasore or dyngo-4a for 30 minutes, in a dose-range, then added pseudovirus. In parallel, we performed infection with mock pseudovirus, which served as a negative control. After 72 hours, we quantified pseudoviral uptake. Infected cells express Green Fluorescent Protein (GFP). We viewed cells with a microscope equipped with epifluorescence, and counted green (infected) cells. We used Hoechst 33342 to stain the nuclei and we calculated the ratio of infected cells to uninfected cells.
4
Lentiviral Transduction Protocol for HEK293T Cells
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Packaging cells (HEK293T) were transfected with a 3‐plasmid system. For transfections, 12 μg pCMVΔR8.2, 6 μg pHIT G, and 12 μg plasmid DNA of interest (pCMV-GFP and pCMV-NRN1-GFP) were combined with DMEM (without phenol red) and subsequently mixed with 24 μl Lipofectamine Plus (Invitrogen) to a final volume of 160 μl (mixture A). Twenty microliters of Lipofectamine LTX (Invitrogen) were mixed with 140 μl DMEM (without phenol red) and incubated for 10 min (mixture B). After incubation, mixtures A and B were combined, incubated for 30 min at RT, and finally added to HEK293T cells, which were seeded the previous day in 10 ml high glucose DMEM into a 10 cm dish. Twenty‐four hours later, lentiviral supernatants were collected and filtered (0.45 μm pore size) for the subsequent infection of target cells (Mel JuSo). Harvested pCMV-NRN1-GFP supernatants were mixed with 1 volume Lenti Concentrator (Origene, cat# TR30025) for every 4 volumes supernatant. Following incubation at 4 °C for 6 h, viral particles were harvested through centrifugation at 3 500 g, 4 °C for 25 min and resuspended in 6 ml. The concentrated viral solutions were then added to the cells of interest for infection. The infected cells were incubated for 16 h, and the medium was subsequently changed to remove remaining virus particles.
5
Lentiviral Plasmids and Pseudoparticle Production
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Lentiviral plasmids encoding shRNA were obtained from Sigma-Aldrich. Each plasmid was transformed into One Shot Stbl3 chemically competent cells (Invitrogen) and purified by ZymoPURE plasmid Maxiprep kit (Zymo research). Lentiviral pseudoparticles were obtained after plasmid transfection of 293FT cells using Lipofectamine 2000 (Invitrogen) or TurboFectin 8.0 Transfection Reagent (Origene). To prepare Vpx-VLPs, 293T cells were co-transfected by Lipofectamine 2000 or TurboFectin 8.0 Transfection Reagent with the 5 mg pMDL-X, 2.5 mg pcRSV-Rev, 3.5 mg X4-tropic HIV Env, and 1 mg pcVpx/myc, as described previously with some modifications37 (link),38 (link). The medium was replaced after 6-12 h with fresh media with IX Viral boost (Alstem). The lentivirus or Vpx-VLPs containing media was harvested 72 h after transfection and concentrated 80 times using Lenti-X concentrator (Takara Clontech) or Lenti Concentrator (Origene). LV particles were then resuspended in RPMI 1640 media without serum and stored at −80°C before use. Virus titer was determined by using Jurkat T cells and Lenti-X GoStix Plus (Takara Clontech).
6
Lentiviral Particle Production and Titration
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Lentiviral plasmids encoding shRNA were obtained from Sigma-Aldrich. Each plasmid was transformed into One Shot Stbl3 chemically competent cells (Invitrogen) and purified by ZymoPURE plasmid Maxiprep kit (Zymo research). Lentiviral pseudoparticles were obtained after plasmid transfection of 293FT cells using Lipofectamine 2000 (Invitrogen) or TurboFectin 8.0 Transfection Reagent (Origene). To prepare Vpx-VLPs, 293T cells were co-transfected by Lipofectamine 2000 or TurboFectin 8.0 Transfection Reagent with the 5 μg pMDL-X, 2.5 μg pcRSV-Rev, 3.5 μg X4-tropic HIV Env, and 1 μg pcVpx/myc, as described previously with some modifications37 (link),38 (link). The medium was replaced after 6–12 h with fresh media with 1X Viral boost (Alstem). The lentivirus or Vpx-VLPs containing media was harvested 72 h after transfection and concentrated 80 times using Lenti-X concentrator (Takara Clontech) or Lenti Concentrator (Origene). LV particles were then resuspended in RPMI 1640 media without serum and stored at −80°C before use. Virus titer was determined by using Jurkat T cells and Lenti-X GoStix Plus (Takara Clontech).
7
Lentiviral Transduction Protocol
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Recombinant lentiviruses were produced by co-transfection of Lenti-X 293T cells (1 × 106 cells in gelatin-coated 10 cm cell culture dish) with lentiviral transfer construct (1.2 pmol), psPAX2 packaging plasmid (1.2 pmol; Addgene # 12260), and pMD.2G envelope plasmid (0.7 pmol; Addgene #12259) using the Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific). Transfection was performed according to the manufacturer’s optimized protocols. Lentiviruses were harvested 3 days post-transfection, filtered through 0.45 μm-pore size PES filters, and concentrated 100 times using Lenti Concentrator (OriGene). The lentiviral titer concentration, determined by Lentivirus Titer Kit HIV-1 p23 Elisa Assay (OriGene), was ~2 × 108 TU/mL.
Lentiviral transduction was carried out with 1 × 105 host cells in 24-well cell culture plate and 10 multiplicities of infection (MOIs) of lentivirus in the presence of 8 μg/mL of polybrene. Lentivirus was removed after overnight incubation, and fresh cell culture media was added. Two days post-transduction, expression of the fluorescent proteins (eGFP, mTurquoise2, and mCherry) were verified using an EVOS fluorescence microscope (Thermo Fisher Scientific). Transduced cells were passaged in culture as bulk preparation for functional assays.
Lentiviral transduction was carried out with 1 × 105 host cells in 24-well cell culture plate and 10 multiplicities of infection (MOIs) of lentivirus in the presence of 8 μg/mL of polybrene. Lentivirus was removed after overnight incubation, and fresh cell culture media was added. Two days post-transduction, expression of the fluorescent proteins (eGFP, mTurquoise2, and mCherry) were verified using an EVOS fluorescence microscope (Thermo Fisher Scientific). Transduced cells were passaged in culture as bulk preparation for functional assays.
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