BMSCs were isolated from mouse compact bone and cultured.46 (link) Briefly, mice were euthanized, and long bones were dissected. The epiphyses were cut off, and the bone cavities were washed. The compact bones were excised into chips of 1–2 mm3, cultured in a 6 cm2 cell culture dish and digested by collagenase II (Worthington). Finally, the bone chips were cultured at 37 °C for 3 days and digested by trypsin (Gibco) for passage. Passage 3–5 cells were used for experiments unless otherwise described. For the generation of BMSCs that overexpress CUL4B, the pcDNA3-CUL4B vector or empty pcDNA3 vector was transfected into BMSCs using Lipo3000 reagent (Thermo).
Lipo3000 reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Lipo3000 reagent is a laboratory product designed for the analysis of lipids. It provides a reliable and efficient method for the quantification and characterization of lipid compounds in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Si nc, by RiboBio (2 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Penicillin streptomycin, by Boster Bio (2 mentions)
Dual luciferase reporter system, by Promega (2 mentions)
M1000, by Tecan (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lncrna gm44593 overexpression plasmid, by GenePharma (2 mentions)
Mir 29b mimics and inhibitor, by GenePharma (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Worthington (1 mentions)
Pcdna3, by GenePharma (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Ges 1, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Solarbio (1 mentions)
Hgc 27, by American Type Culture Collection (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
51 protocols using lipo3000 reagent
1
Isolation and Culture of Mouse BMSCs
2
Overexpressing Cx43 in RPMI-8226 Cells
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The RPMI-8226 cells were transfected with control plasmids (pcDNA3.1) or overexpression plasmids (pcDNA3.1-Cx43). Both the pcDNA3.1 or pcDNA3.1-Cx43 plasmids were purchased from Shanghai GenePharma, Co., Ltd. (Shanghai, China). Before transfection, cells were cultured in 12-well plates till they reached 70% confluency. Opti-MEM medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Lipo 3000 reagent (Thermo Fisher Scientific, Inc.) were used. After transfection for 48 h, cells were collected for subsequent analysis.
3
Regulation of Gastric Cancer Cells by HOXA10-AS
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Human gastric cancer cells HGC-27 and AGS and normal gastric mucosal epithelial cell GES-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 (Sartorius, Göttingen, Germany) containing 10% fetal bovine serum (Solarbio, Beijing, China) at 37°C. Briefly, cells were grown to 80% confluency and detached using Trypsin-EDTA (0.25%, Thermo Fisher, Waltham, MA, USA) for subculture. Culture medium was replaced every 24 hours.
HOXA10-AS was cloned into pcDNA3.1 (HOXA10-AS) for overexpression. siRNAs against HOXA10-AS (si-HOXA10-AS#1, #2, and #3) and HOXA10 (si-HOXA10) and negative controls (si-NC) were purchased from Ribobio (Guangzhou, China). Cells were transfected with si-NC, si-HOXA10-AS (#1, #2, and #3), empty vector, HOXA10-AS, or HOXA10-AS in combination with si-HOXA10 using Lipo3000 reagent (Thermo Fisher) following the manual. After 48-72 hours, cells were harvested for subsequent assays.
HOXA10-AS was cloned into pcDNA3.1 (HOXA10-AS) for overexpression. siRNAs against HOXA10-AS (si-HOXA10-AS#1, #2, and #3) and HOXA10 (si-HOXA10) and negative controls (si-NC) were purchased from Ribobio (Guangzhou, China). Cells were transfected with si-NC, si-HOXA10-AS (#1, #2, and #3), empty vector, HOXA10-AS, or HOXA10-AS in combination with si-HOXA10 using Lipo3000 reagent (Thermo Fisher) following the manual. After 48-72 hours, cells were harvested for subsequent assays.
4
Isolation and Manipulation of Synovial Fibroblasts
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Primary synovial fibroblasts isolated from C57/BL6 mice were seeded on six-well plates at 3 × 105 cells/well (approximately 90% confluence). Cells (primary mouse synovial fibroblasts or SW982 cells [HTB-93 – ATCC]) were transfected with pCMV-vector or pCMV-RGS12 or with shControl, shRGS12, or shKIF2A on the following day with Lipofectamine 3000 reagent (Thermo Fisher Scientific) according to the instructions. Cells were harvested 24–48 h after transfection. For stable transfection, synovial fibroblasts were seeded at 2 × 106 cells per well in a six-well plate and transfected with pCMV-vector or pCMV-RGS12 plasmids using Lipo3000 reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were treated with 0.4 mg/mL geneticin (G418; Thermo Fisher Scientific) every other day for 1 week until G418-resistant colonies had formed. Stably transfected cells were thereafter maintained in complete medium (DMEM supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% FBS) containing 0.4 mg/mL G418.
5
Transfection of GBM Cell Lines
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Two human GBM originated cell lines, U87 and U251, were purchased from ATCC. Cells were cultured in DMEM medium supplied with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Cells were transiently transfected with pcDNA3-NKD1 plasmids (YSY Biotech, Nanjing, China) using pcDNA3-vector as negative control [18 (link)] by Lipo3000 reagent (Thermo Fisher Scientifics, Pittsburgh, USA).
6
Modulation of SNHG12 and WWP1 in LSCC
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The LSCC cell line AMC-HN-8 was purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in 90% DMEM (high-glucose) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin. Cells in culture dishes were placed in a 37°C incubator with 5% CO2 atmosphere. SNHG12 siRNA (si-SNHG12: 5′-GUGCUGCAAUCAACUUUAAUU-3′) [15 (link)], WWP1 siRNA (si-WWP1: 5′-GGAGGCGCUUAUAUGUAAU-3′) and control siRNA (si-NC: 5′-UUGUACUACACAAAAGUACUG-3′) [16 (link)] were purchased from RiboBio (Guangzhou, China). miR-129-5p inhibitor (anti-miR-129-5p) and its negative control (anti-miR-NC) and miR-129-5p mimic and its control (miR-NC) were obtained from GenePharma (Shanghai, China). The cDNA encoding WWP1 was amplified by PCR and then cloned into the vector pcDNA3.1 (Invitrogen, CA, USA), generating the vector pcDNA-WWP1. The empty pcDNA3.1 vector was used as a control. Lipo3000 reagent (Thermo Fisher Scientific, Waltham, USA) was used to assist transfection according to the manufacturers’ protocols. The transfection efficiency was assessed and subsequent assays were performed 48 h after transfection.
7
Selective TERRA Knockdown Using LNA Gapmer
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TERRA was masked by an LNA gapmer mediated approach as previously described [41 (link)]. The LNA gapmer contains phosphorothioate backbone modifications indicated by “*” in the sequence below.
The position of the LNA modifications and the sequence of the LNA gapmer are designed by Qiagen. At first, U2OS cells were transfected with siNC or siRBMX siRNAs. 24 h post the siRNA transfection, cells were then transfected with LNA gapmer by using Lipo3000 Reagent (Thermo Fisher Scientific) and incubated for 24 h. TERRA or control antisense LNA gapmer includes sequences 5′-T*A*A*C*C*C*T*A*A*C*C*C*T*A*A*C-3′ or 5′-C*A*C*G*T*C*T*A*T*A* C*A*C*C*A*C-3′, respectively.
The position of the LNA modifications and the sequence of the LNA gapmer are designed by Qiagen. At first, U2OS cells were transfected with siNC or siRBMX siRNAs. 24 h post the siRNA transfection, cells were then transfected with LNA gapmer by using Lipo3000 Reagent (Thermo Fisher Scientific) and incubated for 24 h. TERRA or control antisense LNA gapmer includes sequences 5′-T*A*A*C*C*C*T*A*A*C*C*C*T*A*A*C-3′ or 5′-C*A*C*G*T*C*T*A*T*A* C*A*C*C*A*C-3′, respectively.
8
DUSP26 Overexpression Assay in U251 and U87 Cells
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U251 and U87 cells were seeded in six-well plates with a density of 70% and cultured overnight. DUSP26 overexpression plasmid (Origene #RC200202) corresponding empty vector were transfected into U251 and U87 cells by using Lipo3000 reagent (Thermo Fisher #L3000015), as per instruction manual. Briefly, 5µg plasmids were diluted in 250 µl OptiMEM and then mixed with 2.5 µl of P3000 reagent. Then, the mixture was added to cultured U251 and U87 cells. The cells were harvested after 48 h, the mRNA and protein levels of DUSP26 were analyzed using quantitative PCR (qPCR) and Western blot.
9
Overexpression and Knockdown of MALAT1, LIN28A, and Nox4
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MALAT1 and coding sequences of LIN28A and Nox4 were cloned into the pcDNA3.1 vector (V79020, ThermoFisher, Waltham, MA, USA) for overexpression of MALAT1 (oe-MALAT1), LIN28A (oe-LIN28A) and Nox4 (oe-Nox4). siRNA (10 nM) was selected for transient transfection thanks to its easy generation and introduction into cells with high efficiency. Specially, si-RNAs against MALAT1 (si-MALAT1), LIN28A (si-LIN28A) and Nox4 (si-Nox4) and scrambled negative control siRNAs (si-NC) were purchased from RiboBio (Guangzhou, China). HK-2 cells were transfected with vector, oe-MALAT1, si-MALAT1, oe-LIN28A, si-LIN28A, oe-Nox4, si-Nox4, si-LIN28A+vector, si-LIN28A+oe-Nox4, si-MALAT1+vector, si-MALAT1+oe-LIN28A or si-NC using the Lipo3000 reagent (L3000015, ThermoFisher) following the manual. Cells were harvested at 48 hours after transfection for subsequent assays. In some assays, cells were harvested and treated with NG or HG. For in vivo knockdown of MALAT1, viral vector-based shRNA with high transduction efficiency and long-term effect was used. Specially, the shRNA against MALAT1 (sh-MALAT1, Sigma) and scrambled shRNA (sh-NC, Sigma) were inserted into the pLKO.1 lentiviral vector (10878, Addgene, Watertown, MA, USA), sh-MALAT1 and sh-NC lentiviral particles were packaged in HEK-293T cells.
10
Overexpression and Silencing of HCG4 in A549 Cells
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The cDNA encoding full-length HCG4 was synthesized (RiboBio) and subcloned into the XhoI and EcoRI sites of the pcDNA3.1 vector (Invitrogen). Myc-RIG-I and HA-Ub-K63 were purchased from MiaoLing Biology (Wuhan, China). For silencing of HCG4 expression, HCG4-specific siRNAs.
(siHCG4:5′-GAAGCUAUGUUUGGAAUUATT-3′; non-targeting control siRNA: 5′-UUCUCCGAACGUGUCACGUTT-3′) were purchased from Gene Pharma Co. (Shanghai, China). Transfection plasmids or siRNAs at the indicated concentrations were transfected into A549 cells using Lipo3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).
(siHCG4:5′-GAAGCUAUGUUUGGAAUUATT-3′; non-targeting control siRNA: 5′-UUCUCCGAACGUGUCACGUTT-3′) were purchased from Gene Pharma Co. (Shanghai, China). Transfection plasmids or siRNAs at the indicated concentrations were transfected into A549 cells using Lipo3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).
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