Transfection reagent

Manufactured by Santa Cruz Biotechnology

Sourced in United States, China

Transfection reagent is a chemical compound used to facilitate the introduction of foreign genetic material, such as DNA or RNA, into cells. It aids in the uptake and expression of the introduced genetic material within the target cells.

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SiRNA transfection reagent (167 citations) ShRNA Plasmid Transfection Reagent (10 citations) Plasmid transfection reagent (5 citations) ShRNA transfection reagent (4 citations) ShRNA Plasmid Transfection Reagent (sc-108061) (4 citations) Transfection reagents and kits (2 citations) Lipofectamine 2000 transfection reagent (2 citations) Transfection reagent (sc-29528 (2 citations) RNAiMax Transfection Reagent (2 citations) Santa Cruz siRNA Transfection Reagent (2 citations)

87 protocols using transfection reagent

Modulating Caveolin-1 Expression in Cancer Cells

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CAV1 was depleted using a pool of three target-specific 20–25 nt siRNA (Santa Cruz Biotechnology). Cancer cells were transfected in 6-well culture plates, at 60–80% confluence, with siRNA CAV1. Cells were also transfected with a scr siRNA in parallel as controls. For each transfection, cells were treated for 5 h with 2.4 μM of siRNA in transfection medium (Santa Cruz) containing 0.5 μL cm⁻² of transfection reagent (Santa Cruz).
CAV1 was amplified in cancer cells using CRISPR activation plasmid (h, Santa Cruz). Cancer cells were transfected in 6-well culture plates, at 60–80% confluence, with CRISPR Activation Plasmid CAV1. For each transfection, cells were treated for 5 h with 0.9 ng μL⁻¹ of CRISPR Activation Plasmid CAV1 in transfection medium (Santa Cruz) containing 0.5 μL cm⁻² of transfection reagent (Santa Cruz).
After incubation with siRNA CAV1 or CRISPR Activation Plasmid CAV1, complete media was added and the cells were incubated for 48 h. CAV1 downregulation or upregulation was evaluated 48 h post-transfection by western blotting.
Further experiments, to determine the effects of CAV1 depletion or amplification on HER2 stability at the cell membrane, were performed at 48 h post-transfection.

Pereira P.M., Sharma S.K., Carter L.M., Edwards K.J., Pourat J., Ragupathi A., Janjigian Y.Y., Durack J.C, & Lewis J.S. (2018). Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy. Nature Communications, 9, 5137.

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TGFβ RII siRNA Transfection in U-373MG Cells

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In this study, TGFβ RII siRNA, scrambled control (nonspecific siRNA as the negative control), and transfection reagent were supplied by Santa Cruz Biotechnology Inc. The U-373MG cells were cultured in a 6-well culture plate (2×10⁵ each well) in serum and antibiotic-free RPMI1640 medium. On the other hand, untreated cells were considered as the controls. Transfection was carried out in line with the manufacturer’s guidelines.
The siRNA transfection medium (Santa Cruz Biotechnology, USA) was used to separately dilute the transfection reagent and TGFβ RII siRNA. After mixing the solution, it was incubated at room temperature for 30 minutes. After rinsing the cells with PBS, the mixture was added to each well of transfection medium (800 μL). Following incubation at 37°C for 6 hours inside a CO₂ incubator, RPMI-1640 medium consisting of 20% fetal bovine serum was added. Under the mentioned conditions, the cells were incubated. Finally, cell harvest was carried out at 48 hours following transfection for further analysis.

Aval F.O., Amiri S.A., Azadmehr A., Oladnabi M., Saadat P., Ebrahimi H., Baradaran B., Mansoori B., Pourabdolhossein F., Torabian P, & Hajiahmadi M. (2018). Gene Silencing of TGFβRII Can Inhibit Glioblastoma Cell Growth. Asian Pacific Journal of Cancer Prevention : APJCP, 19(9), 2681-2686.

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Transfection of Astrocytes with siRNA

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Twenty-four hours prior to transfection with TLR3, Syk, Hrs, and STAM siRNA, 2 × 10⁵astrocytes per well were seeded in a 6-well plate in the antibiotic-free normal growth medium. For each transfection, 40 or 80 pmol of the specific siRNA was added to 8 μl of the Transfection Reagent (Santa Cruz Biotechnology) to obtain Transfection Reagent mixture according to the manufacturer's instructions. Following 45 min incubation of the mixture at room temperature, cells were washed with Transfection Medium (Santa Cruz Biotechnology) and then the Transfection Reagent mixture was overlaid onto the washed cells. After 7 h incubation, a normal growth medium containing 2 times the normal serum was added to the cells without removing the transfection mixture. Astrocytes were cultured for 48 h or 72 h from the beginning of the transfection, and the efficiency of each of the siRNA transfection was affirmed by western blotting (see Figures 2(d), 3(e), and 4(d)).

Mielcarska M.B., Bossowska-Nowicka M., Gregorczyk-Zboroch K.P., Wyżewski Z., Szulc-Dąbrowska L., Gieryńska M, & Toka F.N. (2019). Syk and Hrs Regulate TLR3-Mediated Antiviral Response in Murine Astrocytes. Oxidative Medicine and Cellular Longevity, 2019, 6927380.

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Investigating p21 and p53 Regulation

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All small interfering RNA (siRNA) reagents including transfection reagents were obtained from Santa Cruz biotechnology (Santa Cruz, CA, USA). Cells were transfected with non-target siRNA (siRNA-A), used as negative control, p21 siRNA and p53 siRNA. The experiments were carried out following the manufacturer’s standard procedures. Total protein was extracted, and Western blot analysis was performed.

Hrgovic I., Doll M., Kleemann J., Wang X.F., Zoeller N., Pinter A., Kippenberger S., Kaufmann R, & Meissner M. (2016). The histone deacetylase inhibitor trichostatin a decreases lymphangiogenesis by inducing apoptosis and cell cycle arrest via p21-dependent pathways. BMC Cancer, 16, 763.

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Notch1 Signaling Modulates Osteogenesis

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Notch1 siRNA, scrambled siRNA and transfection reagents were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody against bone ALP was purchased from Abcam (San Francisco, CA). Antibodies against Notch1, NICD1, β-actin, phosphorylated NF-κB p65 (Ser536) and total NF-κB p65 were purchased from Cell Signaling, Inc. (Beverly, MA). Medium 199 was purchased from Lonza (Walkersville, MD). Recombinant Jagged1 and cytokine ELISA kits were purchased from R&D System (Minneapolis, MN). Jagged1 ELISA kit was purchased from Uscn Life Science Inc. (Germany). NF-κB p65 DNA-binding activity assay kit was purchased from Active Motif (Carlsbad, CA). OxLDL was purchased from Biomedical Technologies Inc. (Stoughton, MA). SN50 and BAY11-7082 were purchased from Enzo Life Sciences Inc. (Farmingdale, NY). GSI1 (z-leu-leu-nle-CHO) was purchased from Calbiochem (San Diego, CA). DAPT, LPS (E. coli 0111:B4) and other chemicals and reagents were purchased from Sigma-Aldrich Chemical Co (St Louis, MO).

Zeng Q., Song R., Ao L., Xu D., Venardos N., Fullerton D.A, & Meng X. (2014). Augmented Osteogenic Responses in Human Aortic Valve Cells Exposed to oxLDL and TLR4 Agonist: A Mechanistic Role of Notch1 and NF-κB Interaction. PLoS ONE, 9(5), e95400.

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Transient Knockdown of JunD and PRDX3 in Prostate Cancer

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The transient knock-down of JunD or PRDX3 protein in PC3 and DU145 cells were performed using previously described methods [29 (link)]. In brief, PC3 and DU145 cells were transfected with 60 nM of JunD, PRDX3, or control siRNA using transfection reagents (Santa Cruz Biotechnology) following manufacturer’s recommendations. Seventy-two hours after transfection, the knock-down of JunD or PRDX3 expression in PC3 and DU145 were confirmed by Western blot analysis and then subjected to several functional analyses.

Elliott B., Millena A.C., Matyunina L., Zhang M., Zou J., Wang G., Zhang Q., Bowen N., Eaton V., Webb G., Thompson S., McDonald J, & Khan S. (2019). Essential role of JunD in cell proliferation is mediated via MYC signaling in prostate cancer cells. Cancer letters, 448, 155-167.

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Comprehensive Analytical Techniques for Apoptosis

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TRIzol reagent and apoptosis/necrosis kits were purchased from Invitrogen Co. (Grand Island, NY). AhR pharmacological activators and inhibitors were purchased from Toronto Laboratories Research (Toronto, Canada). RNA isolation and RT-PCR kits were obtained from Applied Biosystems® (Foster City, CA). Acrylamide/bisacrylamide 30% (29:1) and Western blot assay reagents were purchased from Bio-Rad Laboratories (Hercules, CA). Antibodies against target proteins and the manufacturing companies are listed in Suppl. Tab. 1. Interference RNA, transfection reagents and kits were ordered from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Al-Dhfyan A., Alaiya A., Al-Mohanna F., Attwa M.W., AlAsmari A.F., Bakheet S.A, & Korashy H.M. (2022). Crosstalk between aryl hydrocarbon receptor (AhR) and BCL-2 pathways suggests the use of AhR antagonist to maintain normal differentiation state of mammary epithelial cells during BCL-2 inhibition therapy. Journal of Advanced Research, 50, 177-192.

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siRNA Knockdown of Transcription Factor in Human MDMs

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Human MDMs were transfected with siRNA for TF or control siRNA, using transfection reagents (all from Santa Cruz Biotechnology). The efficiency of siRNA knockdown was measured by real-time PCR of TF mRNA expression. Briefly, 10⁶ CD14⁺ MDMs were resuspended in 500 µl of transfection medium, and transfected with siRNA (6 pmoles). After 6 h, an additional 250 µl of 2X RPMI complete medium was added and cells were cultured overnight in a 24-well plate. The next day MDMs were infected with H37Rv, as outlined above, and after 3 days culture supernatants were collected and IL-10 production was measured by ELISA and apoptosis was determined as described below.

Venkatasubramanian S., Tripathi D., Tucker T., Paidipally P., Cheekatla S., Welch E., Raghunath A., Jeffers A., Tvinnereim A.R., Schechter M.E., Andrade B.B., Mackman N., Idell S, & Vankayalapati R. (2015). TISSUE FACTOR EXPRESSION BY MYELOID CELLS CONTRIBUTES TO PROTECTIVE IMMUNE RESPONSE AGAINST Mycobacterium tuberculosis INFECTION. European journal of immunology, 46(2), 464-479.

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Investigating Dasatinib and Rapamycin Signaling

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Dasatinib and Rapamycin were purchased from Selleck Chemical (Shanghai, China). Primary antibodies against phospho-Src (Tyr416), Src, phospho-mTOR (Ser2448), mTOR, phospho-PI3K (Tyr458), PI3K p85, phospho-Akt (Thr308), Akt, phospho-FoxO1 (Ser256), phospho-FoxO3a (Ser253), phospho-4E-BP1 (Thr37/46), 4E-BP1, Cdk2, Cdk4, Cdk6, and Alexa Fluor 555 conjugated antibody were obtained from Cell Signal Technologies (Shanghai, China). Primary antibodies against CDK inhibitor proteins including p16, p19, p21, p27, and β-tubulin were purchased from Santa Cruz Biotechnology (Shanghai, China). Antibodies against phospho-p70S6K (Thr389/412), p70S6K, were purchased from SAB Signalway (College Park, MD, USA). The Secondary HRP- or FITC- conjugated antibodies, si-mTOR, si-Src, and transfection reagents were purchased from Santa Cruz Biotechnology (Shanghai, China).

Chen B., Xu X., Luo J., Wang H, & Zhou S. (2015). Rapamycin Enhances the Anti-Cancer Effect of Dasatinib by Suppressing Src/PI3K/mTOR Pathway in NSCLC Cells. PLoS ONE, 10(6), e0129663.

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Modulating NK Cell Expansion via siRNA

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Freshly isolated human PBMC or mouse spleen cells were transfected with siRNA for IL-21 or control siRNA, using transfection reagents (all from Santa Cruz Biotechnology). The efficiency of siRNA knockdown was measured by real-time PCR of human or mouse IL-21 mRNA expression. Briefly, 10⁶ cells were resuspended in 500 µl of transfection medium and transfected with siRNA (6 pmoles). After 6 h, an additional 500 µl of 2X RPMI-1640 complete medium was added, and cells were cultured overnight in a 24-well plate. The next day, cells were washed and stimulated with either Ag85a or ESAT6, or kept in medium alone as a control. Expansion of memory like NK cell subpopulations was determined after 5 days.

Venkatasubramanian S., Cheekatla S., Paidipally P., Tripathi D., Welch E., Tvinnereim A.R., Nurieva R, & Vankayalapati R. (2016). IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis. Mucosal immunology, 10(4), 1031-1042.

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