To produce lentivirus, HEK293T cells were cultured in 10-cm dishes and transiently transfected with 9 μg lentiviral plasmid pLV-ER-GFP (Cat# 80069, Addgene, a gift from Pantelis Tsoulfas), 8 μg pCMV-dR8.91, and 1 μg PMD2.G packaging plasmids using 25 μl TransIT®-LT1 Transfection Reagent (Cat# MIR 2306, Mirus). After 72 h of transfection, supernatant was filtered through 0.45 μm filters, concentrated using Lenti-X concentrator (Cat# VC100, ALSTEM) at 4°C overnight, and centrifuged at 1,500 × g for 30 min at 4°C to collect virus pellets. The virus pellets were resuspended in cold culture medium for storage at −80 °C for transduction of cells.
Plv er gfp
Manufactured by Addgene
Sourced in United States
The PLV-ER-GFP is a plasmid that expresses green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) of eukaryotic cells. It can be used to visualize the ER network in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Transit lt1 transfection reagent, by Mirus Bio (3 mentions)
15 μ slide 8 well chambers, by Ibidi (1 mentions)
Lck gfp, by Addgene (1 mentions)
Tagrfp, by Evrogen (1 mentions)
Bafilomycin a1 b1793, by Merck Group (1 mentions)
Pmturquoise2 peroxi, by Addgene (1 mentions)
Compound c, by Merck Group (1 mentions)
5 protocols using plv er gfp
1
Lentiviral Production and Concentration
2
Visualizing intracellular organelles and RFP-NS1-2
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LLC-MK2 cells were seeded in 15 μ-slide 8-well chambers (Ibidi, Germany) and transfected 1 day prior to imaging. Cells were transfected with intracellular markers for the plasma membrane (LCK-GFP; Addgene plasmid #61099), endoplasmic reticulum (pLV-ER GFP; Addgene plasmid #80069), Golgi apparatus (pLV-Golgi GFP; Addgene plasmid #79809), and mitochondria (HyPer-dMito; Evrogen). Control wells received TagRFP (Evrogen), while experimental wells received either full-length RFP-NS1-2, RFP-NS1-2(Δ157), or RFP-NS1-2(Δ176). Cells were imaged 24 h posttransfection on the DeltaVision LIVE high-resolution deconvolution microscope (GE Healthcare) using the 60×/1.4 Plan-Apo NA oil objective (Olympus), and acquired using a pco.edge sCMOS_5.5 camera. Images were acquired and deconvolved in SoftWoRx software. After the images were deconvolved, they were further processed in FIJI (ImageJ) to adjust for brightness/contrast and pseudocoloring (82 (link)).
3
Lentiviral Vector Production Protocol
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To produce lentivirus, HEK293T cells were cultured in 10-cm dishes and transiently transfected with 9 μg lentiviral plasmid pLV-ER-GFP (Addgene, 80069, a gift from Pantelis Tsoulfas), 8 μg pCMV-dR8.91, and 1 μg PMD2.G packaging plasmids using 25 μL TransIT-LT1 Transfection Reagent (Mirus, MIR 2306). After 72 h of transfection, supernatant was filtered through 0.45 μm filters, concentrated using Lentivirus Precipitation Solution (ALSTEM, VC100) at 4°C overnight, and centrifuged at 1,500x g for 30 min at 4°C to collect virus pellets. The virus pellets were resuspended in cold DMEM for storage at −80°C for transduction of cells.
4
Mitochondrial Dynamics in Cell Lines
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BEAS-2B, A549, HeLa–Parkin, and MEF cells were maintained in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum (FBS; JR Scientific Inc., Woodland, CA, USA). PINK1 KO MEFs were kindly provided by Dr. Jongkyeong Chung (Seoul National University, Seoul, Republic of Korea) [50 (link)]. Cell lines stably expressing mt-Keima or Keima were generated via infection with a lentivirus produced by using a pLVX-mtKeima lentiviral construct [23 (link)].
pLV-ER GFP (#80069), pLV-Golgi eGFP (#79809), and pmTurquoise2-Peroxi (#36203) were obtained from Addgene (Watertown, MA, USA). A mitochondrial YFP-expressing plasmid (pLESIP-mitoYFP) was generated by subcloning mitoYFP from pcDNA3-mitoYFP (provided by Dr. Gyesoon Yun, Ajou University, Suwon, Republic of Korea) into the pLESIP vector. CCCP (C2759), berberine (14050), bafilomycin A1 (B1793), and compound C (171260) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
pLV-ER GFP (#80069), pLV-Golgi eGFP (#79809), and pmTurquoise2-Peroxi (#36203) were obtained from Addgene (Watertown, MA, USA). A mitochondrial YFP-expressing plasmid (pLESIP-mitoYFP) was generated by subcloning mitoYFP from pcDNA3-mitoYFP (provided by Dr. Gyesoon Yun, Ajou University, Suwon, Republic of Korea) into the pLESIP vector. CCCP (C2759), berberine (14050), bafilomycin A1 (B1793), and compound C (171260) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
5
Lentivirus Production Protocol
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To produce lentivirus, 293T cells were cultured in 10-cm dishes and transiently transfected with 9 μg lentiviral plasmid pLV-ER-GFP (Addgene #80069, a gift from Pantelis Tsoulfas), 8 μg pCMV-dR8.91, and 1 μg PMD2.G packaging plasmids using 25 μl TransIT®-LT1 Transfection Reagent (Mirus #MIR 2306). After 72 h of transfection, supernatant was filtered through 0.45 μm filters, concentrated using Lentivirus Precipitation Solution (ALSTEM #VC100) at 4°C overnight, and centrifuged at 1,500 × g for 30 min at 4°C to collect virus pellets. The virus pellets were resuspended in cold DMEM for storage at −80°C for transduction of cells.
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