Katanin p60 is encoded by the gene katanin catalytic subunit A1 (KATNA1; GenBank Accession no. 007044). The pcDNA3.1 and pcDNA3.1/KATNA1 plasmids were designed and constructed by Chongqing Weisiteng Biomedical Science and Technology Co., Ltd. (Chongqing, China). MDA-MB-231 and MCF-7 cells were seeded in 6-well plates at a density of 1.5×105 cells/well, and transfected with pcDNA3.1 or pcDNA3.1/KATNA1 when the cell confluence reached 50–70%. Two cell lines were transfected under the same condition. A total of 1800 µl Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) was added to each well, followed by a mixture of 2 µg plasmid DNA, 6 µl X-tremeGENE transfection reagent (Roche, Basel, Switzerland) and 200 µl Opti-MEM, the control group was cultured only with Opti-MEM. Following an incubation of 5 h at 37°C, cells were collected at different time points for subsequent studies. The transfection efficiency was detected by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
Opti mem
Manufactured by Roche
Sourced in Switzerland, Germany, United States
Opti-MEM is a cell culture medium developed by Roche. It is designed to support the growth and maintenance of various cell lines in a reduced serum environment. Opti-MEM provides a balanced salt solution and essential nutrients to promote cell viability and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (6 mentions)
X tremegene transfection reagent, by Roche (3 mentions)
Polybrene, by Merck Group (3 mentions)
Opti mem media, by Thermo Fisher Scientific (2 mentions)
X tremegene hp, by Roche (2 mentions)
X tremegene sirna transfection reagent, by Roche (2 mentions)
Sirna oligonucleotide, by Thermo Fisher Scientific (1 mentions)
Polybrene sc 134220, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
X tremegene hp transfection reagent, by Roche (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Shrna clones, by Merck Group (1 mentions)
Mission lentiviral packaging mix, by Merck Group (1 mentions)
Plenti giii cmv gfp 2a puro backbone, by Applied Biological Materials (1 mentions)
Dotap, by Roche (1 mentions)
Facsaria 2, by BD (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
X tremegene reagent, by Roche (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Polybrene, by Beyotime (1 mentions)
17 protocols using opti mem
1
Overexpression of Katanin p60 in Breast Cancer Cells
2
Knockdown of Autophagy and mTOR in Macrophages
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Knockdown of Atg5 or mTOR in macrophages was performed using siRNA oligonucleotides targeting the cDNA sequence of human Atg5 and mTOR according to the manufacturer's protocols (Invitrogen Life Technologies, Inc., Carlsbad, CA, USA). The cells were incubated in 35 mm dishes and transfected with siRNA. An irrelevant 21 nucleotide siRNA (GenePharma Co., Ltd., Shanghai, China) was used as the negative Control. First, 10 μL of siRNA (20 μM) was mixed with 100 μL Opti-MEM media (Invitrogen Life Technologies, Inc., Carlsbad, CA, USA). In addition, 10 μL X-tremeGene Transfection Reagent (Roche, Basel, Switzerland) was mixed with 100 μL Opti-MEM. Then, the two samples were mixed together and combined for 20 min. The mixtures were added to each Petri dish containing fresh medium and incubated for 6 h at 37°C in the dark. Medium was then changed to antibiotic-free FBS-loaded RPMI 1640 medium for 48 h at 37°C in the dark.
Atg5 siRNA Duplexes
Atg5 siRNA Duplexes
S1 (sense, GCAGUGGCUGAGUGAACAUTT, antisense, AUGUUCACUCAGCCACUGCTT)
S2 (sense, CCAUCAAUCGGAAACUCAUTT, antisense, AUGAGUUUCCGAUUGAUGGTT)
S3 (sense: GCUUCGAGAUGUGUGGUUUTT; antisense: AAACCACACAUCUCGAAGCTT)
S1 (sense, CCACCCGAAUUGGCAGAUUTT, antisense, AAUCUGCCAAUUCGGGUGGTT)
S2 (sense, GCAAAGAUCUCAUGGGCUUTT, antisense, AAGCCCAUGAGAUCUUUGCTT)
S3 (sense, CCAAGAUACCAUGAACCAUTT, antisense, AUGGUUCAUGGUAUCUUGGTT)
3
Transfection and viral transduction protocol
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OptiMem (31985070, Life Technologies) and Lipofectamine RNAiMAX (13778075, Life Technologies) were used to complex and transfect 20 µM miR-128 mimic, anti-miR-128 or control mimics (C-301072-01 and IH-301072-02, Dharmacon, Lafayette, CO, USA) into cells. OptiMem and Xtreme Gene HP (06366236001, Roche Life Science, Penzburg, Germany) were used to transfect pJM101 neomycin L1 reporter plasmid into HeLa cells. Cells were transduced with high titer virus using polybrene (sc-134220, Santa Cruz Biotechnology, Dallas, TX, USA) and spinoculation (800 g at 32 °C for 30 min). Transduced cells were then selected and maintained using 3 µg/mL puromycin.
4
TIAM1 Overexpression in Adipose Pericytes
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TIAM1 overexpression was assayed using a human TIAM1 ORF mammalian expression plasmid (RG220233, OriGene). At 24 hours prior to transfection, CD146+ adipose pericytes at passages 3–8 were seeded in 12-well plates at a density of 5 × 104 cells per well. Transfections were performed at 60% confluence; 1 μg of TIAM1 plasmid or control plasmid was mixed with 3 μL of Roche Xtreme gene HP transfection reagent in 100 μL of Opti-MEM and incubated at room temperature (RT) for 30 minutes. The DNA/transfection reagent mixture was then added in drops to wells (42 (link)). qRT-PCR and ICC were used to measure TIAM1 gene expression and to confirm the efficacy of the plasmid.
5
PDPN Knockdown in TPC1 Cells
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TPC1 cells were transfected with siRNA (final concentration 30 nM) targeting human PDPN, (siPDPN, 5′-CGAAGACCGCUAUAAGUCUTT-3′; Life Technologies, Ambion, USA) and a universal negative control siRNA (Sigma-Aldrich, USA) using Lipofectamine 2000 (Life Technologies, Invitrogen, USA) in Opti-MEM (Roche, Switzerland) medium, according to the recommended protocols. The efficiency of PDPN gene inhibition was evaluated 48 h after transfection by RT-qPCR, Western blotting and immunofluorescence. The experiment was repeated four times.
6
Silencing ATF-4/CHOP in Mesenchymal Stem Cells
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The MSCs were seeded in 6-well plates and cultured in serum-free medium for 24 h, and then transfected with siRNA for rat ATF-4/CHOP and non-target siRNA (scramble siRNA) using X-tremeGENE siRNA transfection reagent (Roche, Mannheim, Germany) according to the manufacturer's instructions. The siRNA sequences for ATF-4 and CHOP were 5′-AUCGAAGUCAAACUCUUUCAGGUCC-3′ and antisense, 5′-GGACCUGAAAGAGUUUGACUUCGAU-3′; and 5′-GGAAGAACUAGGAAACGGA-3′ and antisense, 5′-UCCGUUUCCUAGUUCUUCC-3′, respectively. The siRNAs were dissolved in diethylpryocarbonate (DEPC)-treated water and diluted to 0.2 µM with 250 µl Opti-MEM (obtained from Invitrogen, Carlsbad, CA, USA) for 10 min. A total of 10 µl of X-tremeGENE siRNA transfection reagent (Roche) was also diluted with 250 µl Opti-MEM for 20 min. The siRNA and transfection reagent were then mixed (500 µl) and blended gently for 20 min and added to the cell plate with 2 ml culture medium. The MSCs transfected with the siRNAs were cultured for 48 h and then subjected to OGD conditions as described above. The cells were harvested for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis to determine the silencing rate, and for use in subsequent experiments.
7
TWIST1 Expression Modulation in Cells
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For TWIST1 overexpression, lentiviral plasmids encoding full‐length wide‐type TWIST1 with a pLenti‐GIII‐CMV‐GFP‐2A‐Puro backbone (Applied Biological Materials Inc., Vancouver, BC, Canada) were used. For TWIST1 knockdown, two shRNA clones (#TRCN0000020541 and #TRCN0000020542; Sigma‐Aldrich; subsidiary of Merck KGaA: St. Louis, MO, USA) were selected with pLKO.1‐puro Luciferase shRNA plasmid (#SHC007; Sigma‐Aldrich) as a control. Plasmids were mixed with MISSION® Lentiviral Packaging Mix (#SHP001; Sigma‐Aldrich) before added to a mixture of transfection reagent Fugene 6 (#11814443001; Roche, Basel, Switzerland) and OptiMEM. After 10–15 min’ incubation at room temperature, they were added to 293T cells seeded in the 6‐cm dishes. For infection, virus‐containing supernatants were harvested 48 and 72 h after transfection, filtered, and added to selected cells, together with polybrene (Sigma‐Aldrich). Twenty‐four hours after infection, cells were treated with puromycin at a proper concentration decided by their respective puromycin kill curve.
8
Caspase-1 Knockdown in THP-1 Macrophages
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Knockdown of caspase-1 in THP-1 macrophages was performed according to the manufacturer’s protocols using siRNA oligonucleotides targeting the cDNA sequence of human caspase-1 (General Biosystems Inc., Anhui, China). To this end, 10 μL of siRNA (20 μM) was mixed with 100 μL Opti-MEM media (Invitrogen Life Technologies). Separately, 10 μL of X-tremeGene Transfection Reagent (Roche, Basel, Switzerland) was mixed with 100 μL Opti-MEM. The two samples were combined, and 20 min later the mixture was added into individual THP-1 macrophage cultures on a 35-mm dish containing fresh medium, and incubated for 6 h at 37 °C in the dark. Medium was then changed to antibiotic-free, FBS-containing RPMI 1640 medium for 48 h at 37 °C in the dark. Caspase-1 siRNA Duplexes S1 (sense: CCUGUGAUGUGGAGGAAAUTT, anti sense: AUUUCCUCCACAUCAXAGGTT), S2 (sense: UGGAAGACUCAUUGAACAUTT, anti sense: AUGUUCAAUGAGUCUUCCATT), S3 (sense: GAAGACUCAUUGAACAUAUTT; and anti sense: AUAUGUUCAAUGAGUCUUCTT) were used. A siRNA with an irrelevant nucleotide sequence was used as negative control.
9
Cell Stimulation Protocol for Cytokine Secretion
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For cell stimulation, PBMCs were seeded at 3 × 105 cells/well and murine DCs were seeded at 2 × 105 cells/well in 100 µL growth medium in a 96-well flat-bottom plate. Cells were stimulated with CpG ODN 2216 at 1 µM without a transfection reagent as stimulation control. For stimulation with ORNs, RNA40 was applied at 5 µg/mL, and RNA63, RNA66 and RNA28 ORNs were applied at 10 µg/mL. The stimulation mixture was prepared as follows: RNA stimuli were combined with 50 µL Opti-MEM and 1.5 µL DOTAP (Roche, Mannheim, Germany) per well. Samples were incubated for 5 min at room temperature, then 50 µL of the medium was added without serum and used to stimulate cells at 100 µL. Supernatants were harvested 20 h post stimulation (h p. s.) and cytokine secretion was determined by ELISA analysis.
10
Retroviral Transduction of LMO1 in 293T Cells
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The LMO1 cDNA (CDS region of LMO1 transcript, NM_001270428.1) was amplified and cloned into the pMSCV-IRES-GFP retrovirus vector. Retroviral vector (1000 ng) was co-transfected into 293T cells (200,000 cells) seeded into six-well plate 24 h before transfection with the packaging plasmid pMD-MLV (250 ng) and the envelope plasmid VSV-G (250 ng) using FuGENE 6 reagent (Roche) and Opti-MEM. Approximately 500,000 cells were transduced with 1 mL retrovirus media in the presence of polybrene (8 μg/ml: Millipore) for 2 h incubation followed by toping up of 1 mL fresh medium. The cells that express GFP were then sorted by flow cytometry using the BD FACSAria II (BD Biosciences).
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