We performed intravitreal injections in the first week after diabetes induction. Intraperitoneal anesthesia with mixture of ketamine and xylazine (Sigma-Aldrich Corp. St. Louis, MO, USA.) was administered to the three groups and iodophor disinfection was conducted around the eyes subsequently. A thirty-Gauge needle (Becton, Dickinson and Company. Franklin Lakes, NJ, USA.) was inserted using a Hamilton microinjector (Hamilton Company, Reno, NV, U.S.A) toward the optic nerve at 1 mm outside of the limbus under a microscope. The medicine was slowly injected after the needle tip was detected in the pupil area. A volume containing 1 μL of 20 μmol/L GLUT1 siRNA and 1 μL of transfection reagent was intravitreally injected into the GLUT1 siRNA treatment group, whereas a volume containing 1 μL of 20 μmol/L non-targeted siRNA and 1 μL of transfection reagent (Invitrogen, Waltham, MA, USA) was intravitreally injected into the scrambled siRNA and diabetic scrambled siRNA groups. The injection was conducted in both eyes and repeated twice a week until nine injections were completed.
Transfection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Transfection reagent is a laboratory product used to introduce nucleic acids, such as DNA or RNA, into cells. It facilitates the uptake of these molecules by the cells, enabling researchers to study gene expression, knockdown, or other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase assay system, by Promega (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Dual luciferase reporter assay kit, by Promega (4 mentions)
Dual luciferase reporter assay system, by Promega (4 mentions)
Puromycin, by Merck Group (4 mentions)
Saos 2, by American Type Culture Collection (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
R0531, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Cell culture media, by Thermo Fisher Scientific (2 mentions)
Progesterone, by Merck Group (2 mentions)
α tubulin, by Merck Group (2 mentions)
Irdye 680lt goat anti rabbit igg, by LI COR (2 mentions)
On target pooled sirnas, by Thermo Fisher Scientific (2 mentions)
Irdye 800cw goat anti mouse igg 926 32210, by LI COR (2 mentions)
Alexa fluor 488 goat anti mouse a 11029, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit a11008, by Thermo Fisher Scientific (2 mentions)
92 protocols using transfection reagent
1
Intravitreal GLUT1 siRNA Injection
2
Silencing TRAP1 in Glioblastoma Cells
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shRNA sequences targeting human TRAP1 and the empty vector were purchased from Shanghai GenePharma Co., Ltd. The TRAP1 shRNA sequence was as follows: 5'-CCGGCAGAGCACTCACCCTACTATGC-TCGAGCATAGTAGGGTGAGTGCTCTGTTTTTG-3'. Plasmids were transduced into cells using transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, SHG44 cells grown in 6-well plates were transfected with 4 µg TRAP1-shRNA or empty vector control and 6 µl transfection reagent was added to each well. Cells were harvested 24 h later and samples were further analyzed by WB analysis.
3
Silencing Ae. albopictus Genes via siRNA
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A set of siRNAs targeting the different Ae. albopictus genes were selected to perform siRNA reverse transfection assays in C6/36 cells, including Chaperonin-60kD, ch60 partial mRNA (NCBI Accession: XM_001661714.1), Enolase phosphatase e-1 partial mRNA (NCBI Accession: XM_001657643.1) and Spermatogenesis associated factor partial mRNA (NCBI Accession: XM_001654630.1) and were purchased from Sigma-Aldrich Corp., St Louis, MO, USA. The siRNAs were diluted from 100 μM stock concentration to working concentrations of 0.1, 1.0, 10, 30 and 50 nM with DharmaFECT Cell Culture Reagent (DCCR) (Thermo Scientific) and transfection reagent (Dharmafect-1) to make up a final volume of 100μL per well. After which, siRNAs were incubated for 30 minutes at room temperature. The positive control was replaced with DCCR. 1.2×106 C6/36 cells in 400 μL were seeded into 24-well plate together with the siRNA-transfection mixture. After 48 hours post-transfection, the siRNA-transfected cells were subjected to CHIKV-infection with a M.O.I. 10 for 24 h.p.i. The viral supernatant was harvested and viral plaque assays were performed.
4
Recombinant Protein Expression and Purification
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Fc-6X-His- and AP-6X-His-tagged recombinant proteins were expressed by transient transfection of HEK293T cells grown in media containing 10% Ultra-Low IgG fetal bovine serum (Invitrogen; Carlsbad, CA) using linear polyethylenimine (PEI) transfection reagent (Thermo Fisher Scientific). For 15 cm plates, 32 μg of plasmid DNA and linear PEI (Cf=40 μg/ml) was added to 3.2 ml Opti-MEM (Invitrogen), vortexted briefly, incubated for exactly 10 minutes at room temperature and added dropwise onto cells. Culture media was harvested 6 days post transfection. The amount of Fc- and AP-tagged proteins in the media was quantified as described previously (Wojtowicz et al., 2007 (link)). For stripe assays, 6X-His-tagged proteins were purified using TALON metal affinity resin (Clontech Laboratories; Mountain View, CA) and quantified using the Bradford assay as described previously (Wojtowicz et al., 2004 (link)).
5
Transfection and Proliferation Assay of gEEC
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The chi-let-7b-5p mimic and inhibitor, as well as the corresponding NC mimic and inhibitor, were bought from the Guangzhou Ruibo biological company. Immortalized gEECs were obtained as previously described [61 (link)] The cells were transfected once they had fused to 50%. In brief, chi-let-7b-5p mimic was diluted with serum-free medium Opti, mixed with transfection reagent (ThermoFisher Scientific, Waltham, MA, USA), incubated at room temperature for 20 min, added to the appropriate well drop by drop, and collected after 48 h for follow-up operation. Following transfection, 10 μL CCK8 (AbMole Bioscience Inc., Houston, TX, USA) was added to each well at the appropriate time in order to study the impact of chi-let-7b-5p on the proliferation of gEEC. Incubation was then continued for another two hours. The absorbance of gEEC at 450 nm was measured using a Thermo Scientific Microplate Reader (ThermoFisher Scientific, Waltham, MA, USA).
6
Recombinant Hepcidin Protein Production
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Hepcidin CDS sequences were inserted into a plasmid with zebrafish β-actin promoter at EcoRI and BamHI sites. EGFP and mCherry DNA sequence was inserted into CDS regions following 5′ terminal of propeptide and 3′ terminal of mature peptide by PCR and In-Fusion Cloning, respectively. These plasmids were transfected into Chinese hamster ovary (CHO) cells using transfection reagent (Thermo Fisher). The hepcidin mature sequences were inserted into pHis-TEV using NcoI and XhoI enzyme digesting sites. The recombinant plasmid with His tag sequence at the 5′ terminal of mature hepcidin sequence can be transformed into E. coli to produce hepcidin protein controlled by T7 promoter. All the recombinant plasmids were verified by sequencing.
7
Transfection of Primary Lung Fibroblasts
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Transfections of primary lung fibroblasts were performed using Lipofectamine 2000 (for DNA) or RNAiMAX (for siRNA) Transfection Reagent (Thermo Fisher Scientific, catalog nos. 11668027 and 13778150). Forty-eight hours later, transfected cells were analyzed by Western blots or treated with EVs for uptake assay.
8
Luciferase Assay for Gene Expression
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For luciferase assays, 2 × 104 cells/well were seeded in 48-well plate and transfected with the pmirGLO-Rab25-WT, pmirGLO-Rab25-MUT and a negative control using transfection reagent (R0531, Thermo, USA). At 48 h post-transfection, cells were lysed and luciferase activity was examined using the dual-luciferase reporter assay system (E1910, Promega, USA) according to the manufacturers’ instructions. All experiments were performed in triplicate and the results were expressed as firefly luciferase activity normalized to Renilla luciferase activity.
9
Lipofectamin 3000-Mediated Plasmid Transfection
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The plasmid was purchased from (GenePharma Co. Ltd., Shanghai, China). Transfection reagent was purchased from (Thermo Fisher Scientific, Shanghai, China). After the cells have reached 60-70% growth, Lipofectamin 3000 was added to 100 µl of serum-free medium, and the pcDNA and P3000 (1:1) were added to 100 µl of serum-free medium, both were mixed and incubated for 15 min at room temperature. After incubation, the mixture is added to each well (12-well plate) with 800 ul of serum-free medium and then 200 ul of P3000-Lipofectamin3000-pcDNA mixture is added to each well and incubation is continued at 37°C in a constant temperature incubator; after 24-36 h of transfection, subsequent experiments can be carried out.
10
Flow Cytometry for CRISPR Genome Editing
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The flow cytometry protocol was described previously (15 (link)). Briefly, 1.8 × 105 293-SC1 cells/well were seeded in 12-well plates on day 1, and transfected with Cpf1 and crRNA expression plasmids by the Transfection Reagent (TurboFect, Thermo Scientific) on day 2. Fresh medium was added to the transfected 293-SC1 cells on day 3. Cells were harvested for flow cytometry or genomic DNAs isolation on day 4. loxP-STOP-loxP-mG/FnCpf1 study was performed as described previously (16 (link)).
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