Silencing of HIF-1α expression in SHED was achieved by transfection of premade siRNA (siHIF-α:4390824, negative control: 4390843, Thermo Scientific) with LipofectamineTM 3000 (Thermo Scientific) according to the manufacturer’s instructions. Briefly, the cells were seeded in 6-well plates for 70 ∼ 90% density. After overnight incubation, lipofectamineTM 3000 reagent – siRNA complexes were prepared in Opti-MEMTM Reduced Serum Medium (Thermo Scientific). Subsequently, the fresh culture medium was changed and siRNA-lipid complexes were added into the cells. siRNA carrying a no significant sequence was used as a negative control. Transfection efficiency was assessed by western blotting (WB) and immunofluorescence (IF) after 48 h under hypoxia. For identification of different groups, we termed the siRNA-control group as “siControl” and the siRNA-HIF-1α group as “siHIF-1α.” To confirm the function of HIF-1α, we also used HIF-1α inhibitor YC-1 (80 μM, Sigma, MO, United States), which acts by prevention of HIF-1α accumulation in response to hypoxia. Accordingly, we termed the YC-1-control and YC-1-treated groups as “CTR” and “YC-1” groups, respectively.
Opti memtm reduced serum medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Opti-MEMTM reduced serum medium is a cell culture medium formulated to provide reduced serum requirements. It is designed to support the growth and maintenance of a variety of cell lines while minimizing the use of serum.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Plus reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Sigenome smartpool sirna, by Horizon Discovery (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Countess, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Total rna extraction reagent, by Yeasen (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
First strand cdna synthesis kit, by Yeasen (1 mentions)
9 protocols using opti memtm reduced serum medium
1
Silencing HIF-1α in Stem Cells
2
Knockdown of Stem Cell Factors in MDA-MB231 Cells
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siGENOME SMARTpool siRNAs were purchased from Dharmacon as follows: OCT4/POU5F1 (Cat# M-019591-03-0005); SOX2 (Cat# M-011778-00-0005); SOX9 (M-021507-00-0005); Non-Targeting siRNA Control Pool #1 (Cat# D-001206-13-05). MDA-MB231 cells transduced with the SORE6>GFP reporter were transfected with 20 nM siRNAs using Lipofectamine RNAiMAX Reagent (Invitrogen #13778075) in OptiMEMTM Reduced Serum Medium (Thermo Fisher Scientific #31985062). The % SORE6+ cells in the transfected cultures was assessed after 3 days by flow cytometry, using cells transduced with the minCMV>GFP as a gating control.
3
miR-9-5p Regulation of TGF-β1 Signaling
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BEAS-2B cells were transfected with 20 nM of miR-9-5p mimics (Integrated DNA Technologies, Coralville, Iowa, USA) using lipofectamine® RNAiMAX transfection reagent (ThermoFisher Scientific, Cat # 13778075) and Opti-MEMTM reduced-serum medium (ThermoFisher Scientific, Cat # 31985062) according to manufacturer’s instruction. 24 hours post-transfection, cells were treated with TGF-β1 (10 ng/ml) and harvested 16 hours after treatment to quantify mRNA expression by qRT-PCR analysis.
4
Transfection and Patch-Clamp of OtopLa in HEK293 Cells
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We cultured HEK293 cells (ATCC, catalog number ATCC® CRL-2828™, RRID_CVCL_0045) in 35 mm Petri dishes (Fisher Scientific, catalog number 12-556-000) with a medium composed of Opti-MEMTM Medium (GibcoTM, Opti-MEMTM Reduced Serum Medium, Thermo Fisher Scientific, catalog number 51985034), 10% fetal bovine serum (GibcoTM, Thermo Fisher Scientific, catalog number 16140063), and 100 U/mL penicillin–streptomycin (GibcoTM, Thermo Fisher Scientific, catalog number 15140122). We maintained the cells in a tissue culture incubator at 37 °C with 5% CO2. Cells were passaged once a week, and the culture medium was replaced every 3 days.
We co-transfected pcDNA3-OtopLa or pcDNA3-OtopLaE638A together with pcDNA3-GFP (5 : 1) into HEK293 cells using LipofectamineTM 3000 transfection reagent (InvitrogenTM, Thermo Fisher Scientific, catalog number L3000008). Cells were treated with 0.25% trypsin (Thermo Fisher Scientific, catalog number 15090046) 24 h after transfection and were subsequently plated onto coverslips coated with poly-l -lysine (Sigma-Aldrich, catalog number P4707) for the patch-clamp recording. The same procedure was used to co-transfect pCS2+MT-OtopLa or pCS2+MT-OtopLaE638A.
We co-transfected pcDNA3-OtopLa or pcDNA3-OtopLaE638A together with pcDNA3-GFP (5 : 1) into HEK293 cells using LipofectamineTM 3000 transfection reagent (InvitrogenTM, Thermo Fisher Scientific, catalog number L3000008). Cells were treated with 0.25% trypsin (Thermo Fisher Scientific, catalog number 15090046) 24 h after transfection and were subsequently plated onto coverslips coated with poly-
5
Transfection of FAH-GFP Construct in M1 Cells
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Human fibroblastoid M1 cell line (34 (link)) was grown in complete DMEM supplemented with 10% (v/v) fetal bovine serum, 0–1 mg/ml streptomycin, and 100 units/ml penicillin at 37 °C with 5% CO2 in incubator chamber. To maintain and amplify cells in a proper confluence, they were counted by means of an automated cell counter (CountessTM, Life Technologies) that performs cell count and viability calculations (from alive, dead, and total cells) using Trypan blue staining.
Transfection of a plasmid carrying the FAH-GFP constructs (pCMV6-AC-GFP) in M1 cells was performed using X-tremeGENETM HP DNA transfection reagent (Roche) following the manufacturer's instructions. The first day, 500,000 cells per P100 dish and plasmid construction were seeded in complete DMEM and allowed to attach for 24 h. On the second day, complete medium was replaced by 4 ml of Opti-MEMTM reduced serum medium (Gibco, Ref. 31985). After 2 h, the medium was discarded, 1 ml of transfecting solution was poured drop by drop, and 3 ml of Opti-MEMTM were added to completely cover the cells. After 4 h of incubation, 3 ml of complete DMEM was added. The following day, the medium was changed for complete DMEM supplemented with 2 mg/ml of G418, GeneticinTM (Thermo Fisher, catalog no. 11811031) for clone selection.
Transfection of a plasmid carrying the FAH-GFP constructs (pCMV6-AC-GFP) in M1 cells was performed using X-tremeGENETM HP DNA transfection reagent (Roche) following the manufacturer's instructions. The first day, 500,000 cells per P100 dish and plasmid construction were seeded in complete DMEM and allowed to attach for 24 h. On the second day, complete medium was replaced by 4 ml of Opti-MEMTM reduced serum medium (Gibco, Ref. 31985). After 2 h, the medium was discarded, 1 ml of transfecting solution was poured drop by drop, and 3 ml of Opti-MEMTM were added to completely cover the cells. After 4 h of incubation, 3 ml of complete DMEM was added. The following day, the medium was changed for complete DMEM supplemented with 2 mg/ml of G418, GeneticinTM (Thermo Fisher, catalog no. 11811031) for clone selection.
6
Investigating Nanoparticle Transfection Mechanisms
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For pDNA transfection, cells were seeded in 24-well plates at a density of 5 × 104 cells per well and waited for adhesive overnight at 37 °C with 5% CO2. Before the transfection, the original culture media were replaced by 300 μL Opti-MEMTM Reduced Serum Medium (Gibco, Waltham, MA, USA) for 1 h. For pDNA transfection, 50 μL of nanoparticles solution containing 500 ng pDNA was added to the wells and gently mixed with the Opti-MEM. As for siRNA-GAPDH transfection, 50 μL of nanocomplex solution was prepared by calculation of the concentration of siRNA in final culture media to be 40 nM. Cells were then incubated with the nanocomplex for 6h under the condition of 37 °C with 5% CO2, before being replaced with the fresh original complete media.
Cell transfection was repeated with changing the culture environment in 4 °C, or with the addition of either 100 μg/mL chloroquine (Aladdin, Shanghai, China), 5 mg/mL Methyl-β-cyclodextrin (MβCD) (Aladdin, Shanghai, China), 10 mg/mL Chlorpromazine (Aladdin, Shanghai, China) or 10 mg/mL Genistein (Aladdin, Shanghai, China) to investigate the mechanism of endocytosis and endosomal escape of the nanoparticles. Cells transfected with the addiction of various inhibitors or incubated under 4 °C were analyzed by flow cytometer.
Cell transfection was repeated with changing the culture environment in 4 °C, or with the addition of either 100 μg/mL chloroquine (Aladdin, Shanghai, China), 5 mg/mL Methyl-β-cyclodextrin (MβCD) (Aladdin, Shanghai, China), 10 mg/mL Chlorpromazine (Aladdin, Shanghai, China) or 10 mg/mL Genistein (Aladdin, Shanghai, China) to investigate the mechanism of endocytosis and endosomal escape of the nanoparticles. Cells transfected with the addiction of various inhibitors or incubated under 4 °C were analyzed by flow cytometer.
7
Lentiviral Vector Production and Transduction
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Lentivirus production was adapted from ref. (92 (link)). HEK293FT cells were grown to ~80% confluency in 10-cm or six-well plates, for 10 mL or 2 mL final viral media harvest, respectively, and transfection reagents were scaled according to seeding area. For a 10-cm plate, 3.5 – 4E6 cells were seeded and incubated for 24 h (37 °C, 5% CO2). Transfection reagents were prepared in Opti-MEMTM reduced serum medium (Gibco) and performed using 94.2 µL Lipofectamine 2,000 (ThermoFisher), 103.6 µL PLUSTM reagent (ThermoFisher), 8.2 µg psPAX2, 5.4 µg pMD2.G, and 10.7 µg construct DNA. The mixture was incubated at room temperature for 5 min and gently added to the HEK293FT cells for 4 h incubation (37 °C, 5% CO2). The medium was then replaced with prewarmed harvest media (DMEM 30% FBS). Forty-eight h after the start of the transfection, the lentivirus supernatant was collected and filtered (0.45 µm). Transductions were conducted directly at the time of lentivirus harvest or freshly thawed from frozen aliquots. 0.5 to 1 mL of virus media and polybrene (1 µg/mL) were added to cells seeded in a six-well plate in 1 to 1.5 mL of growth media. Cells were spinfected at 2,250 rpm, 1 h, room temperature (25 °C), and incubated overnight (37 °C, 5% CO2). Twenty-four h posttransduction, cells were selected by puromycin (2 µg/mL) for 48 h.
8
Characterization of Doxorubicin Treatment Effects
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Penicillin-Streptomycin (10,000 U/mL) antibiotics (Gibco, 15140122), Bicinchoninic acid (BCA) Protein Assay Kit (Pierce, 23227), Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, 11965092), Fetal Bovine Serum (FBS) (Gibco, 10270106), and Opti-MEMTM Reduced Serum Medium (Gibco, 31985062) were purchased from ThermoFisher Scientific (Waltham, MA, USA). Hexadimethrine bromide (polybrene, 107689), puromycin dihydrochloride (>98%, P9620), 4′,6′-diamidino-2-phenylindole (DAPI, 10236276001), and doxorubicin were purchased from Sigma-Aldrich (St. Louis, MI, USA). Nucleic Acid Gel Stain (YeaRed, 10202ES76), Agarose (10208ES76), Cell Counting (CCK-8) Kit (40203ES76), total RNA extraction reagent (Trizol, 10606ES60), first Strand cDNA Synthesis Kit (11121ES60), and Hieff™ qPCR SYBR® Green Master Mix (11203ES08) were purchased from Yeasen Biotech (Shanghai, China). Tris base-acetic acid- EDTA (TAE) buffer (ST716), DNA loading (D0071), and Relilla luciferase reporter kit (RG017) were acquired from the Beyotime company (Shanghai, China).
9
Lentivirus Production and Transduction
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Lentivirus production was adapted from Joung et al., 2017. (link) HEK293FT cells were grown to ~80% confluency in 10-cm or 6-well plates, for 10 mL or 2 mL final viral media harvest respectively and transfection reagents were scaled according to seeding area. For 10-cm plate, 3.5 -4E6 cells were seeded and incubated for 24h (37°C, 5% CO2). Transfection reagents were prepared in Opti-MEM TM reduced serum medium (Gibco) and performed using 94.2 μL Lipofectamine 2000 (ThermoFisher), 103.6 μL PLUS TM reagent (ThermoFisher), 8.2 μg psPAX2, 5.4 μg pMD2.G and 10.7 μg construct DNA. The mixture was incubated at room temperature for 5min and gently added to the HEK293FT cells for 4h incubation (37°C, 5% CO2). The medium was then replaced with prewarmed harvest media (DMEM 30% FBS). 48h after the start of the transfection, lentivirus supernatant was collected and syringed through a 0.45μm filter. Transductions were conducted directly at the time of lentivirus harvest or freshly thawed from frozen aliquots. 0.5-1 mL of virus media and polybrene (1 μg/mL) were added to cells seeded in 6-well plate in 1-1.5 mL of growth media. Cells were spinfected at 2250RPM, 1h, room temperature (25°C) and incubated overnight (37°C, 5% CO2). 24h post-transduction, cells were selected by puromycin (2 μg/mL) for 48h.
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