Gene silencing with siRNA was performed as described (23 (link), 30 (link), 49 (link)). In brief, to deplete α1B/D-ARs from the cell surface, cells were incubated in Nunc six-well plates (Thermo Fisher Scientific) for 3 d in Accell transfection media (GE Dharmacon) with α1B-AR, α1D-AR, or NT (negative control) siRNA (GE Dharmacon) at a concentration of 1 µM. On day 3, cells were centrifuged at 300 × g for 5 min and resuspended in RPMI 1640 (Sigma) supplemented with 10% HyClone FBS from Cytiva (Marlborough, MA), 100 μg/mL penicillin from Invitrogen (Waltham, MA), and 100 μg/mL streptomycin (Invitrogen). Cells were utilized on day 4 for experimentation.
Accell transfection media
Manufactured by Horizon Discovery
Sourced in New Zealand
Accell transfection media is a laboratory product designed to facilitate the delivery of nucleic acids, such as plasmids, siRNA, or mRNA, into cells. It is a specialized cell culture medium that is optimized to enhance the efficiency of transfection, the process of introducing foreign genetic material into cells.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Hyclone fbs, by Cytiva (2 mentions)
Nunc six well plates, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Arginine vasopressin, by Merck Group (1 mentions)
Phentolamine, by Merck Group (1 mentions)
Conivaptan, by Merck Group (1 mentions)
Detection reagents, by Merck Group (1 mentions)
Isotype control igg, by R&D Systems (1 mentions)
Anti ccr2, by R&D Systems (1 mentions)
Anti avpr1a, by Bioss Antibodies (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Phenylephrine, by Merck Group (1 mentions)
Anti cxcr4, by Abcam (1 mentions)
3 protocols using accell transfection media
1
Gene Silencing with siRNA Depletion
2
Receptor Interaction Profiling in Cells
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The antibodies anti-α1B-AR (host: rabbit; catalog#: ab169523), anti-α1D-AR (host: rabbit; catalog#: ab84402), and anti-CXCR4 (host: goat, ab1670) were obtained from Abcam; anti-AVPR1A (host: rabbit; catalog#: bs-11598R) was from Bioss; anti-AVPR1A (host: mouse, catalog#: LS-C126889) and anti-CCR8 (host: goat; catalog#: LS-C187704) were from LifeSpan Biosciences; anti-CXCR1 (host: rabbit, catalog#: PA5-33452) was from Invitrogen; and anti-CCR1 (host: mouse; catalog#: MAB145), anti-CCR2 (host: mouse; catalog#: MAB48607), IgG isotype control (host: rabbit, catalog#: MAB1050), IgG isotype control (host: mouse, catalog#: MAB004), and IgG isotype control (host: goat, catalog#: AB-108-C) were from R&D Systems. CCL2, CCL2325-99, CXCL8, and CXCL12 were purchased from Protein Foundry. Phenylephrine, phentolamine, arginine vasopressin, conivaptan, and poly-L-lysine were purchased from Sigma-Aldrich. AVPR1A siRNA and nontargeting (NT) siRNA and Accell transfection media were purchased from GE Dharmacon. Proximity ligation rabbit, mouse, and goat +/− probes and detection reagents were purchased from Sigma-Aldrich.
3
Depletion of AVPR1A from Cell Surface
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Gene silencing with siRNA was performed as described previously (14 (link), 15 (link)). In brief, to deplete AVPR1A from the cell surface, cells were incubated in Nunc six-well plates (Thermo Fisher Scientific) for 3 d in Accell transfection media (GE Dharmacon) with AVPR1A or NT (negative control) siRNA (GE Dharmacon) at a concentration of 1 μM. On day 3, cells were centrifuged at 300g for 5 min and resuspended in RPMI 1640 (Sigma-Aldrich) supplemented with 10% HyClone FBS from Cytiva, 100 μg/ml penicillin from Invitrogen (Waltham, MA), and 100 μg/ml streptomycin (Invitrogen). Cells were used on day 4 for experimentation.
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