The HEK293T, B16-F10, LO2, Hep3B, Huh7, HepG2, and PLC/PRF/5 cells were all maintained in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum plus streptomycin (100 mg/mL) and penicillin (100 U/mL). The lncRNA-DAW-overexpressing cells were produced by using the retroviral gene delivery method. To generate virus particles, equal amounts of pBABE-lncRNA-DAW plasmid and virus packaging vector pCL-Ampho were co-transfected into HEK293T cells. The supernatant of culture medium was harvested and the virus particles were enriched by 0.45-μm pore size nitrocellulose membranes (Millipore, United States) at day 2. The virus concentration was determined by virus titer assay under the microscope. The virus was diluted to 1 × 106 TU/mL and used for infection. The indicated cell lines were then incubated with the above supernatant plus hexadimethrine bromide (Sigma-Aldrich, United States). Then the retrovirus-infected cells were selected with puromycin (Sigma-Aldrich, United States) at day 4. The virus-infected cells were harvested after antibiotic selection and the relative RNA levels were determined by qRT-PCR. As for the proteasome inhibitor experiment, HepG2 cells were transiently transfected with or without lncRNA-DAW expression plasmid. HepG2 cells were exposed to the proteasome inhibitor MG132 (20 μM) for 6 h before harvest.
0.45 μm pore size nitrocellulose membranes
Manufactured by Merck Group
Sourced in United States
The 0.45 μm pore size nitrocellulose membranes are a lab equipment product used for filtration and separation purposes. The membranes have a pore size of 0.45 micrometers, which allows for the effective retention of particles and molecules above this size. Nitrocellulose is the material used in the construction of these membranes.
Automatically generated - may contain errors
Lab products found in correlation
Hexadimethrine bromide, by Merck Group (3 mentions)
Puromycin, by Merck Group (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using 0.45 μm pore size nitrocellulose membranes
1
Overexpression of lncRNA-DAW in Human Liver Cancer Cells
2
Overexpression of H19 in Colorectal Cancer
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H19 overexpression stable transfectants were constructed using retrovirus-mediated gene delivery method in human colorectal cancer cell lines. For generation of retrovirus, 2 μg expression vector pBABE-H19 or corresponding empty vector were co-transfected with equal amount of packaging vector pCL-Ampho into virus packaging HEK293 cells at 70% confluence in 100 mm culture dish using Lipofectamine 2000 reagent (Invitrogen, USA). After 24 hours post-transfection, the supernatant containing retrovirus were harvested and filtered by 0.45 μm pore size nitrocellulose membranes (Millipore, USA). The human colorectal cancer cell lines, namely HT-29, HCT-116 and SW620 cells, were infected with retrovirus particles together with 8 μg/mL Hexadimethrine bromide (Sigma-Aldrich, USA). After 48 hours post-infection, retrovirus infected cells were selected with puromycin (Sigma-Aldrich, USA) in the concentration of 0.7 μg/mL for HT-29 cells, 2 μg/mL for SW620 cells, and 0.9 μg/mL for HCT-116 cells. To maintain the stable expression of H19 in colorectal cancer cell lines, the infected cells were cultured in the selection medium with puromycin continuously. After antibiotics selection for around 2.5 weeks, the cells was harvested and the relative H19 RNA level was monitored by qRT-PCR.
3
Western Blot Analysis of E1A and GOLPH2
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Cells with different treatments were washed three times with ice-cold PBS, and lysed in buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 100 μg/ml PMSF, 1% TritonX-100) for 30 min at ice. Cells were collected and cell debris was removed by centrifugation. The protein concentration of cell lysates was measured by bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Protein samples were diluted with sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, and then separated by 10% SDS-PAGE and transferred onto 0.45 μm pore size nitrocellulose membranes (Millipore Corp., MA, USA). Then, nonspecific reactivity was blocked with 5% fat-free dry milk in Tween 20-containing Tris-HCL buffered saline overnight at 4°C. The membrane was incubated with primary antibody for E1A and GOLPH2 protein (Santa Cruz Biotech., USA). After incubation in the dark with IR Dye 800 conjugated IgG secondary antibodies (Rockland Inc., UK), immunodetection was performed by using an Odyssey infrared imaging system (LI-COR Biosciences Inc., Lincoln, NE, USA). The same membrane was then used for detecting the expression of GAPDH using primary antibody against human GAPDH (Santa Cruz Biotech., USA).
4
Stable H19 Overexpression in hMSCs
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The H19 overexpression stable hMSCs were generated using retrovirus-mediated gene delivery method as previously described37 (link). To produce retrovirus for infection, 3 μg pBABE-H19 vector or corresponding control empty vector were co-transfected with an equal amount of viral packaging vector pCL-Ampho into HEK293 cells in 100 mm culture dish by using Lipofectamine 2000 transfection reagent (Invitrogen, USA). After transfection, the supernatant containing retrovirus particles were collected and subsequently filtered by 0.45 μm pore size nitrocellulose membranes (Millipore, USA). The hMSCs were infected by the retroviral particles together with 8 μg/mL Hexadimethrine bromide (Sigma-Aldrich, USA). Then, retrovirus infected hMSCs were selected by puromycin (Sigma-Aldrich, USA) in the concentration of 0.5 μg/mL. After antibiotics selection for around 7 days, some cultured cells were collected and the overexpression of H19 was confirmed by qRT-PCR.
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