All lentivirus production was performed as previously described [52 (link)]. Medium was collected from plates 72 hr after transfection, filtered by VIVASPIN (Sartorius, VS2001), concentrated and stored –80°C. hESC and HT-29 cells were infected by lentiviral particles incubated in the growth medium containing and 8μg/ml Polybren (Sigma, 107689) to attached cells, following selection after 24h for several passages for pool isolation.
Polybren
Manufactured by Merck Group
Sourced in United States
Polybren is a cationic polymer used in cell culture applications. It facilitates the adhesion and growth of cells in vitro by improving cell attachment to culture surfaces.
Automatically generated - may contain errors
Lab products found in correlation
Doxycycline, by Merck Group (2 mentions)
Vivaspin, by Sartorius (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Puromycin, by Merck Group (1 mentions)
Mission shrna library, by Merck Group (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Chir99021, by Merck Group (1 mentions)
Pd0325901, by Merck Group (1 mentions)
Mouse embryonic fibroblasts, by Merck Group (1 mentions)
Mitomycin c treated, by Merck Group (1 mentions)
Human recombinant basic fgf, by ProSpec (1 mentions)
6 protocols using polybren
1
Lentiviral Production and Transduction
2
Generating Inducible β-Globin Cell Lines
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To generate cell lines with inducible expression of β-globin sequence variants, we first cloned WT β-blogin cDNA into the pRAR expression vector using restriction-free (RF) cloning76 , followed by cloning of the specific tiles from NucLibA. Lentiviral particles were generated as previously described83 (link): HEK293T cells were transfected with a mixture of pRAR plasmid:pMDL:pVSVG:pRev at the ratio of 1:0.65:0.35:0.25, respectively, using PEI. Medium was collected from plates 48 h after transfection and filtered.
MCF-7 cells were infected by the addition of growth medium containing lentiviral particles and 8 μg/ml Polybren (Sigma). Selection was done by the addition of Puromycin (InvivoGen) at a concentration of 1 μg/ml. Pools of infected cells that survived the selection were used for smFISH; cells were transfected with siRNAs (see above), and 32 h after transfection Doxycycline (Sigma, D9891) was added to a final concentration of 1 µg/ml. Cells were grown for an additional 16 h and were fixed using 4% PFA in PBS.
MCF-7 cells were infected by the addition of growth medium containing lentiviral particles and 8 μg/ml Polybren (Sigma). Selection was done by the addition of Puromycin (InvivoGen) at a concentration of 1 μg/ml. Pools of infected cells that survived the selection were used for smFISH; cells were transfected with siRNAs (see above), and 32 h after transfection Doxycycline (Sigma, D9891) was added to a final concentration of 1 µg/ml. Cells were grown for an additional 16 h and were fixed using 4% PFA in PBS.
3
Lentiviral Transduction of Cells
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Lentiviral particles were from the Sigma-Aldrich MISSION® shRNA library; each shRNA was inserted in a pLKO.1-PURO plasmid (shNon Target, shUBPY #35, shUBPY #39 and shUBPY #67).
Cells were seeded in 96-well plates (#655090, Greiner) at 12000 cells per well in RPMI. After 18 hours, the medium of each well was replaced with RPMI containing 8μg/ml polybren (Sigma-Aldrich). Lentiviral particles were added at a M.O.I of 5 and 72 hours later, 1μg/ml puromycin (Sigma-Aldrich) was added.
Cells were seeded in 96-well plates (#655090, Greiner) at 12000 cells per well in RPMI. After 18 hours, the medium of each well was replaced with RPMI containing 8μg/ml polybren (Sigma-Aldrich). Lentiviral particles were added at a M.O.I of 5 and 72 hours later, 1μg/ml puromycin (Sigma-Aldrich) was added.
4
Silencing Piezo1, GPER, and YAP in Chondrocytes
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siRNA for human Piezo1, GPER and YAP gene was obtained from Jima Gene Company (Shanghai, China). Chondrocytes were seeded in a six-well plate, and when chondrocytes reached 80% confluency, targeting siRNA or scrambled sequence wrapped by lentivirus with titer of 50 nM were added. Polybren (Sigma-Aldrich, St. Louis, USA) was used to facilitate the transfection process.
5
Inducible Pluripotent Stem Cell Generation
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Venus neonatal fibroblasts were harvested using 0.25% Trypsin‐EDTA solution, and 2 × 104 cells were plated per well of 0.1% gelatin‐coated 6‐well plates. Cells were transduced 24 hr after plating using a total MOI of 20 for both viruses (pOSMK and rTA) and 8 μg/mL polybren (Sigma–Aldrich). Forty‐eight hours after addition of lentivirus, the medium was replaced with complete DMEM containing 2 μg/mL doxycycline (Sigma–Aldrich) to induce transgene transcription. Fours days later, the transduced Venus neonatal fibrobalsts were transferred onto mitomycin C–treated (Sigma–Aldrich) mouse embryonic fibroblasts (Millipore). On Day 5, the medium was replaced with piPSC medium containing 10 ng/mL LIF (Millipore) or 20 ng/mL human recombinant basic FGF (Prospec, East Brunswick, NJ), 2i (1 μM PD0325901 [Sigma–Aldrich], 3 μM CHIR99021 [Sigma–Aldrich]), and 2 μg/mL doxycycline (Sigma–Aldrich). On Day 17, colonies were visible and were manually picked to establish clonal lines. The Venus piPSCs were maintained in reduced oxygen (5% O2, 5% CO2 in N2) in a humidified chamber at 38.5°C. Cells were dissociated with 1× TrypLE (Gibco Thermo Fisher Scientific, Waltham, MA), and passaged 1:6 every 7 days.
6
Lentiviral Transduction of Cell Lines
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For production of the replication-defective lentiviral virions, the packaging cell line HEK293T was co-transfected with the packaging plasmid pCMVΔR8.9, the envelope plasmid containing the G-Protein of vesicular stomatitis virus and the transfer vector (pFUGW). 24 The transfer vector includes the reporter gene EGFP driven by an internal ubiquitine-C promoter and the specific shRNA under the control of the human H1 promoter. The pFUGW derivate vectors with the specific C/EBPβ-shRNA inserted were named 'pF-C/EBPβ,' vectors without the shRNA insert were called 'pF-S.' The lentiviral infection of KiJK and Karpas 299 cells (1 × 10 6 cells per ml) was performed in 12-well plates in the presence of Polybren (8 μg/ml; Sigma, St Luis, MO, USA). Cells were centrifuged at 800 g and 32 °C for 90 min. Subsequently the cells were incubated for 24 h at 37 °C in a CO 2 incubator.
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