Platinum E cells were grown to 80–90% confluency in 10 ml of DMEM with 10% FBS and 1% antibiotics in a 100 mm tissue culture dish. Cell were transfected with control vector or retroviral vector containing BATF, IRF4, dCas9-VP64, gRNA-CNS1, gRNA-CNS-25, caSTAT5, and STAT6VT open reading frame by using Lipofectamine 3000 (ThermoFisher Scientific). Eighteen micrograms of retroviral vector, 6 μg pCL-Eco, and 50 μl P3000 were mixed in 500 μl Opti-MEMTM I reduced serum medium (ThermoFisher Scientific), and 50 μl Lipofectamine 3000 was mixed in another 500 μl Opti-MEMTM I reduced serum medium. After combining, this mixture was incubated at room temperature for 15 min. The combined mixture was gently added to the Platinum E cells culture dish. After 16 h, the media containing the retrovirus was collected and replaced with fresh DMEM media for 4 days. The media containing the virus was centrifuged at 1500 r.p.m. for 5 min to remove cell debris. Virus supernatant was used for retroviral transduction or stored at −80 °C for subsequent use.
Opti memtm 1 reduced serum medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Opti-MEMTM I Reduced Serum Medium is a cell culture medium designed to support the growth and maintenance of various cell types while reducing the amount of serum required. It is formulated to provide essential nutrients and growth factors to cells in a reduced serum environment.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Rnase free water, by Thermo Fisher Scientific (1 mentions)
Ahr sirna s1200, by Thermo Fisher Scientific (1 mentions)
Antagomir, by RiboBio (1 mentions)
Antagomir nc, by RiboBio (1 mentions)
Mirna agomir, by RiboBio (1 mentions)
Lipo3000, by Thermo Fisher Scientific (1 mentions)
Nectin4 sirna, by Thermo Fisher Scientific (1 mentions)
Control sirna, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
New born calf serum, by Cambrex (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Heparin, by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (1 mentions)
7 protocols using opti memtm 1 reduced serum medium
1
Retroviral Transduction of Platinum E Cells
2
Culturing and Characterizing Mouse Cancer Cell Lines
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The LLC cells used in this study were the LLC-Mhi cell line (obtained from Dr. Glenn Merlino, NCI), which is a high metastatic subline derived from LLC tumors described previously (Day et al., 2012 (link)). B16F10 cells were purchased from American Type Culture Collection. ARH-77 and ARH-77-mSDC1 cells were kindly provided by Dr. Ralph D. Sanderson (University of Alabama at Birmingham), and RAW264.7 cells by Dr. Raymond P. Donnelly (FDA). LLC, ARH-77, and RAW264.7 cells were all tested negative for mycoplasma (NCI Core facility) and authenticated by STR analysis (IDEXX BioResearch). LLC and B16F10 cells were cultured in RPMI 1640 Medium (LONZA) with heat-inactivated fetal bovine serum (FBS), supplemented with penicillin/streptomycin (1:100) at 37 ˚C, 5% CO2. Culture of LLC cells was carried out under various concentrations of FBS, as indicated in the Figure legends. For LPS stimulation, RAW264.7 cells were cultured in OPTI-MEMTM I reduced serum medium (Thermo Fisher Scientific) for times indicated in the text. LPS transfection was performed using X-tremeGene HP DNA transfection reagent (Roche Applied Science).
3
Evaluating AhR Knockdown Effects
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Negative control siRNA or AHR siRNA (s1200) (both purchased from Invitrogen) was diluted with RNase-free water (Thermo Fisher Scientific), further mixed with Opti-MEMTM I Reduced Serum Medium (Thermo Fisher Scientific) and LipofectamineTM RNAiMAX Transfection Reagent (Thermo Fisher Scientific), and incubated for 20 min at room temperature. HaCaT cells were seeded in 12-well plates and mixed with siRNA-Lipofectamine complex (final siRNA concentration: 10 nM). After 48 h of incubation, cells were treated with DMSO (0.1%), BaP (1 μM) or BAI (25 μM) for 6 h and collected for RNA extraction. The knockdown efficiency of AHR at 48 h after transfection was determined by qRT-PCR and Western blotting.
4
Efficient AAV and miRNA Transduction in Primary Neurons
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For AAV transduction in primary neurons, the viral particles were added to neurobasal medium at a multiplicity of infection of 1 × 106 on the second day of plating. The neurons were analyzed on 14 days in vitro (DIV). The transduction efficiency was observed at > 90%. For miRNA transduction in primary neurons, the neurons were transfected on DIV 12. According to the manufacturer’s instructions, Lipo3000 (L3000008, Invitrogen) was used to transfect 100 nm of miRNA agomir or antagomir into each well of cells using Opti-MEMTM I Reduced Serum Medium (31985062, Thermo Fisher). The cells were examined 48 h after miRNA agomir or antagomir transfection. The miRNA agomir, antagomir, negative control NC agomir, and NC antagomir were purchased from RiboBio (Guangzhou, China).
5
NECTIN4 Knockdown in Cell Lines
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Cells were transfected with control siRNA (Invitrogen, Carlsbad, CA, USA; AM4611) or NECTIN4 siRNA (Invitrogen; s37689) using Lipofectamine RNAiMAX (Invitrogen; 13778075). Briefly, siRNAs were diluted with Opti-MEMTM I Reduced Serum Medium (Thermo Fisher Scientific, Waltham, MA, USA; 31985062), mixed with Lipofectamine, and incubated for 20 min at room temperature. Then, the siRNA-Lipofectamine complexes were mixed with the cells (final siRNA concentration of 10 nM). At 24–72 h after transfection, the cells were harvested and used for further analysis. The knockdown efficiency was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting.
6
Spheroid Formation via siRNA Knockdown
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For the short interfering RNA (siRNA) experiment, adherent cells to achieve 50–70% confluency on the following day were transfected at a final concentration of 30 nM with ON-TARGETplusTM Human non-targeting siRNA (#D-001810-01-20, Dharmacon) or ON-TARGETplusTM SMARTpool Human NAMPT-targeting siRNA (#L-004581-00-0005, Dharmacon) using LipofetamineTM RNAiMAX Regent (#13778-150, Thermo Fisher Scientific) and Opti-MEMTM I Reduced Serum Medium (#11058-021, Thermo Fisher Scientific). On the following day, cells were trypsinized and seeded onto ultra-low attachment plates. 72 h following transfection, spheroids were harvested.
7
HUVEC Culture and Gal-8 Treatment
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Cell culture. In the present study we used primary cultures of HUVECs that were isolated and expanded following published protocol (52) (link) and with respect to the Helsinki Declaration (53) (link) with an informed consent from donors. The study was approved by the Ethical committee of the Faculty of Pharmacy, Comenius University in Bratislava (06/2019). All in vitro experiments were performed using HUVECs at passage 2-3 isolated from 3 donors. Cells were cultured on gelatin-coated dishes in M199 supplemented with 20% heat-inactivated new born calf serum (both from Cambrex, Verviers, Belgium), 150 μg/ml crude endothelial cell growth factor (ECGF), 5 U/ml heparin, 100 IU/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen, Carlsbad, CA, USA) at 37˚C under 5% CO 2 / 95% air atmosphere. Twenty-four hours prior to the experiments the endothelial cell cultures were refreshed with Opti-MEM TM I Reduced Serum Medium (Thermo Fisher Scientific, Waltham, MA, USA). Cell viability, estimated by trypan blue exclusion, was higher than 95% before each experiment. As a positive control, 25 ng/ml of VEGF (R&D Systems, Minneapolis, MN, USA) (54) (link), was used in all experiments. Recombinant human Gal-8 (R&D Systems) was tested at the following concentrations: i) 0.004, ii) 0.02, iii) 0.1, iv) 0.5, and v) 1.0 μg/ml.
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