Virus stocks were prepared by co-transfecting the pLenti-LucDsRed (LT) plasmid with three packaging plasmids, pMDLg/pRRE, CMV-VSVG and RSV-Rev (Addgene, Cambridge, MA, USA), into 293 T cells. Supernatants containing viral particles were harvested 36–48 h later, filtered and centrifuged at 20,000 g for 90 min. Viral titer was determined by the end-point dilution method by counting the number of infected 293 T red cells at 100× magnification under a fluorescence microscope (Nikon, Tokyo, Japan) 96 h after infection. The titer of transducing units (TU) was computed as follows: TU/ml = (the numbers of red fluorescent cells) × (dilution factor)/(volume of virus solution). LLC cells were seeded in 12-well plates and the cells were transduced with an equal amount of LT virus particles. Stably transducted cells were designated as LLC-LT.
Rsv rev
Manufactured by Addgene
Sourced in United States
The RSV-Rev is a lab equipment product. It is designed to perform a core function, but details about its intended use are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pmdlg prre, by Addgene (2 mentions)
Polybrene, by Merck Group (2 mentions)
Vsv g, by Addgene (2 mentions)
Cmv vsvg, by Addgene (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
293t cells, by Takara Bio (1 mentions)
Md2 g, by Addgene (1 mentions)
Gag pol, by Addgene (1 mentions)
Lentiviral helper plasmids, by Addgene (1 mentions)
Psuper gfp vector, by Oligoengine (1 mentions)
Shatg5 lv atg5 rnai 9513 1, by Genechem (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Mission non target shrna control vector, by Merck Group (1 mentions)
8 protocols using rsv rev
1
Lentiviral Transduction of LLC Cells
2
Lentiviral Silencing of Nr5a2 in LLC-SD Cells
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The lentivirus plasmid PLL3.7 (Addgene, USA) was constructed with shRNA specific for Nr5a2 or negative control (GenePharma, China) after enzyme digestion. Recombinant lentiviruses expressing Nr5a2 shRNA or negative control shRNA were obtained by plasmid transformation. Lentivirus was packaged in 293T cell line using the VSVG, pMDLg/pRRE and RSV‐REV (Addgene, USA), as well as Lipofectamine 2000 (Invitrogen, USA). Medium containing lentivirus was collected and filtered through 0.22 μM filter (Millipore, USA) after 48 hours. Fresh filtered virus containing medium was used for LLC‐SD cell transfection or stored at −80 °C for future use. LLC‐SD cells were infected with lentivirus and polybrene (Sigma, USA) added with the final concentration of 8 μg/mL.
3
Lentiviral Transduction of Retinal Ganglion Cells
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Lentivirus was produced using calcium phosphate transfection as described74 (link). One day before transfection 5 × 106 293T cells (Clontech) were seeded onto a 10-cm cell culture plate. The following day cells were transfected with 15 μg MD2.G (Addgene #12259), 6 μg MDL/pRRE (Addgene #12251), 6 μg RSV/rev (Addgene #12253), and 15 μg of the GFP-transfer vector (Addgene #17451)75 (link). At 24 hours post transfection, 10 mM sodium butyrate was added to the cells. Lentivirus was harvested and concentrated at 48 hours post transfection using Lenti-X Concentrator (Clontech). To transduce sorted RGCs, lentivirus with 8 μg/mL Polybrene (Sigma) was added to the cell culture media overnight at 5–10 multiplicity of infection. The next day, the cells were washed with PBS and cultured with N2B27 media.
4
Production and Characterization of SARS-CoV-2 Spike Pseudovirus
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Lenti-X 293 T cells were transfected with Gag/Pol (#12251, Addgene), RSV-Rev (#12253, Addgene), pcDNA3.1(+)-2019-nCoV-spike-P2A-eGFP (#MC_0101087, MolecularCloud), and pCDH-EF-GFP-luc in 24-well plates. After 24 h, 1 × 105 T cells were added to the culture with or without S-BiTE stimulation. Supernatants were collected at 48 h post-transfection. The viral titer was measured by infecting 293-ACE2 cells. Levels of TNF and IFN-γ in the supernatant were analyzed by the CBA assay. The remaining virus-releasing cells were imaged by REVOLVE fluorescent microscopy (ECHO, San Diego, CA, USA) and analyzed by flow cytometry.
5
Lentiviral Knockdown and Overexpression of GDF15
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The GDF15 shRNA target sequence was 5′-TCTCAGATGCTCCTGGTGTTG-3′. A lentiviral pSUPER-GFP vector was purchased from OligoEngine (USA). Lentiviral helper plasmids (PMDL, VSVG and RSV-REV) were obtained from Addgene (Biovector Inc, USA). GDF15-overexpressing lentivirus was obtained from Shanghai Genechem Co., Ltd. Virus supernatant was incubated on target cells for 12 hours with 5 μg/ml polybrene, following the manufacturer’s instructions. Infected cells were selected in puromycin, as optimized for each cell line.
6
Lentiviral Knockdown of S100A8 Gene
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The lentivirus particles of shATG5 LV-ATG5-RNAi (9513-1) were purchased from GENECHEM. The sequence for the synthesized shRNA targeting S100A8 was 5′-CCUGAAGAAAUUGCUAGAGTT-3′. The annealed oligonucleotide fragment was cloned into the lentivirus plasmid PLL3.7 (Addgene, Inc.) to establish the shRNA lentiviral vector. Recombinant lentiviruses expressing S100A8 shRNA or negative control (Shanghai GenePharma Co., Ltd.) were obtained by plasmid transformation. VSVG, pMDLg/pRRE, RSV-REV (Addgene, Inc.) and Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.) were used for lentivirus packaging in 293T cells. The supernatant of the 293T culture was collected and filtered using a 0.22-µm filter (EMD Millipore), followed by a concentration step. Medium containing lentivirus was stored at −80°C for future use.
7
Pseudotyped Lentivirus Neutralization Assay
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Lenti-X 293 T cells were transfected with lentivirus package component plasmids, Gap/pol (#12251, Addgene), RSV-Rev (#12253, Addgene), pCDH-EF-IRFP-luc, and pcDNA3.1(+)-2019-nCoV-spike-P2A-eGFP (#MC_0101087, Molecular Cloud). Supernatants containing lentivirus particles were collected 48 and 72 h post transfection for direct usage or concentration by ultracentrifugation. The viral titer in TU/mL was determined by flow cytometric analysis of the transduced 293-ACE2 cells.
In the virus neutralization assay, S-BiTE was serially diluted to the indicated concentrations in complete Dulbecco’s modified Eagle medium (DMEM). Pseudotyped lentiviral particles were inoculated on 293-ACE2 or A549-ACE2 monolayers in 96-well plates in the presence of 10 μg/mL of polybrene and indicated concentration of S-BiTE, and further incubated at 37 °C for 48 h. For infecting A549-ACE2 cells, a 60-min spin at 2500 rpm at 32 °C was used to improve infection efficiency. IRFP reporter activity was measured using CytoFLEX S (Beckman Coulter). The percentage of infectivity was calculated as the ratio of the IRFP readout in the presence of the fusion protein to the IRFP readout in the absence of the fusion protein. The half-maximal inhibitory concentrations (IC50) were determined using a 4-parameter logistic regression (GraphPad Prism, version 8).
In the virus neutralization assay, S-BiTE was serially diluted to the indicated concentrations in complete Dulbecco’s modified Eagle medium (DMEM). Pseudotyped lentiviral particles were inoculated on 293-ACE2 or A549-ACE2 monolayers in 96-well plates in the presence of 10 μg/mL of polybrene and indicated concentration of S-BiTE, and further incubated at 37 °C for 48 h. For infecting A549-ACE2 cells, a 60-min spin at 2500 rpm at 32 °C was used to improve infection efficiency. IRFP reporter activity was measured using CytoFLEX S (Beckman Coulter). The percentage of infectivity was calculated as the ratio of the IRFP readout in the presence of the fusion protein to the IRFP readout in the absence of the fusion protein. The half-maximal inhibitory concentrations (IC50) were determined using a 4-parameter logistic regression (GraphPad Prism, version 8).
8
Knockdown of PLVAP Expression in In Vitro BRB Models
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For knockdown of PLVAP expression, shRNA lentiviral pLKO.1 constructs (SIGMA/TRC; Sigma-Aldrich, Zwijndrecht, the Netherlands) were used. Control cells were transduced with nontargeting shRNA (MISSION Non-Target shRNA Control Vector; Sigma-Aldrich). Virus particles were generated by co-transfecting the constructs with three packaging plasmids, pMDLg/pRRE, pMD2G, and RSV-Rev (Addgene, Cambridge, MA), into 293T cells. The sequences of PLVAP shRNA and Non-Target shRNA were 5 0 -CC-GGCCCTTTCACACACACTTTCTACTCGAGTAGAAAG-TGTGTGTGAAAGGGTTTTTTG-3 0 and 5 0 -CCGGCAACA-AGATGAAGAGCACCAACTCGAGTTGGTGCTCTTC-ATCTTGTTGTTTTT-3 0 , respectively.
In Vitro BRB Model Cells were isolated as described previously. 6 Three different models were assembled to study endothelial barrier permeability, including i) a bovine retinal endothelial cell (BREC) monolayer, ii) a triple co-culture model with BRECs, pericytes, and astrocytes, and iii) human umbilical vein endothelial cells (HUVECs). In the triple co-culture model, BRECs were seeded on top of the Transwell filter, primary rat astrocytes were seeded on the reverse side of the Transwell filters, and bovine primary pericytes were cultured on the bottom of a 24-well plate in which a Transwell filter was placed, as described previously. 6 Three independent experiments were performed for each model (n ! 11).
In Vitro BRB Model Cells were isolated as described previously. 6 Three different models were assembled to study endothelial barrier permeability, including i) a bovine retinal endothelial cell (BREC) monolayer, ii) a triple co-culture model with BRECs, pericytes, and astrocytes, and iii) human umbilical vein endothelial cells (HUVECs). In the triple co-culture model, BRECs were seeded on top of the Transwell filter, primary rat astrocytes were seeded on the reverse side of the Transwell filters, and bovine primary pericytes were cultured on the bottom of a 24-well plate in which a Transwell filter was placed, as described previously. 6 Three independent experiments were performed for each model (n ! 11).
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