Ramos cells were propagated in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (GIBCO) and antibiotic mixture (Sigma) in a 5% CO2 atmosphere at 37°C. DT40 cells were propagated in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum and 1% chicken serum (GIBCO) and antibiotic mixture (Sigma) in a 5% CO2 atmosphere at 40°C. The AviPack packaging system was utilized for the ASLV-derived virus propagation and pseudotyping with vesicular stomatitis virus protein G (VSV-G) as described in Plachy et al. (2010) (link). HIV-derived vector was produced by 293T cell line co-transfection (X-Treme HP, Roche) with 1 μg of GFP7 vector, 1 μg of psPAX2 (Addgene plasmid # 12260) and 1 μg of pVSV-G (Clontech) in a 6 cm Petri dish. Viral supernatants were collected, filtered through a 0.45 μm SFCA filter and stored at −80°C.
X treme hp
Manufactured by Roche
Sourced in Japan
The X-treme HP is a high-performance laboratory instrument designed for efficient and accurate sample processing. It features advanced technology to ensure reliable and consistent results. The core function of the X-treme HP is to provide a versatile and efficient solution for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Pspax2, by Addgene (2 mentions)
Antibiotic mixture, by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Pvsv g, by Takara Bio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Luciferase assay system, by Promega (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
G418 sulfate, by Merck Group (1 mentions)
Luciferase activity, by Promega (1 mentions)
7 protocols using x treme hp
1
Virus Propagation and Pseudotyping
2
Pseudotyped HIV-1 NL4-3.Luc Virus Production and Infection Assay
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The HIV-1 pNL4-3.Luc.R-E-plasmid [40 (link)], was cotransfected with pVSVG using X-treme HP (Roche) to produce Vpr-negative pseudotyped HIV-1 NL4-3.Luc viruses (HIV-1). The MLV pSra-luc plasmid, which was provided by Dr. Guangxia Gao (Institute of Biophysics, Chinese Academy of Science), was cotransfected with phit60 and pVSVG using X-treme HP. Viruses were collected at 48 and 72 h after transfection. The collected virus was centrifuged for 30 min at 4500×g, and filtered through 0.45 μm filters. The quantity of HIV-1 was assessed with HIV-1 p24 ELISA kit (Advanced BioScience Laboratories). HEK293 cells stably expressing TRIM-HA or knocking down of TRIM11 were seeded in 12-well plates, and infected with different amounts of HIV-1 for 24 h or MLV for 48 h. THP-1 cells stably expressing TRIM11-HA or knocking down of TRIM11 were seeded in 12-well plates in the presence of 100 nM PMA for 48 h, followed by 48 h inoculation with different amounts of HIV-1. Cells were collected and luciferase activity was measured using the Luciferase Assay System (Promega). The luciferase activities were normalized to the protein quantity measured by BCA Protein Assay kit.
3
Plasmid Transfection and siRNA Knockdown Protocols
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The expression plasmids for C-terminally Flag-tagged TβRI or Myc-tagged TβRII [80 (link)], C-terminally haemagglutinin (HA)-tagged p52ShcA [12 (link)] and C-terminal Myc-Smad3D407E [52 (link)] were described. An expression plasmid encoding His-tagged p52ShcA [81 (link)] was a gift from Dr. John Ladbury. The expression plasmids for GFP-tagged β2 adaptin [55 (link)] and GFP-tagged caveolin-1 [82 (link)] were provided by Dr. Ed Leof (Mayo Clinic) and Dr. Martin A. Schwartz (Yale School of Medicine), respectively. Control siRNA and siRNA oligonucleotides targeting mouse or human ShcA [83 (link)] were from Qiagen (Table 1 ). Lentiviral vectors expressing control shRNA or shRNA targeting human and mouse ShcA were from Sigma-Aldrich (Table 2 ). For plasmid transfections, NMuMG, HaCaT, or 293T cells were plated in six-well plates and transfected with Lipofectamine 2000 (Invitrogen) or Xtreme HP (Roche). Five hours after transfection, cells were transferred to fresh medium-containing 10% FBS and incubated for 24–48 h. For siRNA transfections, NMuMG or HaCaT cells were plated in six-well plates and transfected with RNAiMax (Invitrogen). Eight to twelve hours after transfection, cells were transferred to fresh medium containing 10% FBS, cultured for another 12 h, followed by a second siRNA transfection and incubation for an additional 48–72 h.
4
HEK293 Transfection and Luciferase Assay
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The HEK293 cells were seeded in 12-well plates and transfected the following day with the corresponding plasmids using X-treme HP (Roche, Indianapolis, IN). In addition, 50 ng of thymidine kinase (TK)-Renilla luciferase reporter plasmids (pRL-TK) were mixed into each reaction for the normalization of the transfection efficiencies. Twenty-four hours after transfection, dual-specific luciferase reporter gene assays were performed using the Dual-Luciferase Reporter Assay System in accordance with the manufacturer's instructions (Promega, Madison, WI). Firefly luciferase activity was normalized based on the activity of Renilla luciferase.
5
Virus Propagation and Pseudotyping
6
Overexpression and Knockdown of KIAA0101 in ESCC
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In order to overexpress KIAA0101, the full-length KIAA0101 cDNA was cloned into the mammalian expression vector p-EGFP (RiboBio, China) to generate the KIAA0101-overexpressing plasmid. The empty p-EGFP plasmid was used as a control. The plasmids were transfected into the cell lines using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. At 48 h after transfection, G418-sulfate (600 ng/ml for EC9706, and 800 ng/ml for TE1; Sigma-Aldrich; Merck KGaA) was added to the cell culture medium throughout the experimental periods, as previously described (7 (link)). In order to knock down KIAA0101, the ESCC cells were transfected with the pSIREN-Shuttle vector (RiboBio) encoding the KIAA0101-targeted shRNA (5′-GCAACCTGATCACACAAATGA-3′) using Xtreme HP (Roche), according to the manufacturer's instructions. For the transfection of small RNA molecules, ESCC cells were cultured in 6-well plates (2×105 cells per well), and the miR-216a-5p mimic (30 nM), or the miR-216a-5p inhibitor (300 nM), or the negative controls (all were from RiboBio) were transfected into cells using Lipofectamine RNAiMAX (Life Technologies; Thermo Fisher Scientific, Inc.). The cells were used at 48 h after transfection.
7
HIV-1 Vpr+ and Vpr- Pseudovirus Production and Luciferase Assay
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The HIV-1 pNL4-3.Luc.R+E- or pNL4-3.Luc.R-E- plasmids, which were provided by Dr. K. Tokunaga (National Institute of Infectious Diseases, Japan), were cotransfected with pVSVG using X-treme HP (Roche) to produce Vpr-positive or Vpr-negative pseudotyped HIV-1 NL4-3.Luc viruses (HIV-1 Vpr+ and HIV-1 Vpr−). Virus supernatants were harvested and filtered (0.45-µm pore size) at 48 h post-transfection. The viral titers were measured with a p24 ELISA kit (Advanced BioScience Laboratories, Inc). Cell lines carrying the pCDH and pLKO.1-shRNA lentiviral expression vectors were seeded in 48-well plates and infected with different amounts of HIV-1 Vpr−. The cells were collected at 24 h post-infection for the measurement of luciferase activity (Promega), which was normalized by the protein OD of each sample.
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