siRNA Smart pools for TC10, p190RhoGAP, p120RasGAP were purchased from Dharmacon/GE Healthcare (siGenome). Transfections were performed with Oligofectamine 2000 (Invitrogen) for MTLn3 cells and via electroporation, using Amaxa cell line nucleofector kit V (VACA 1003, Lonza, Basel, Switzerland), for MDA-MB-231 cells. To monitor the transfection efficiency, siGLO-Red (Dharmacon) was co-transfected, according to the manufacturer’s protocols. Knockdown was assessed, and subsequent assays were performed at 48 h (MTLn3) or 72 h (MDA-MB-231) after transfection.
Siglo red
Manufactured by Horizon Discovery
Sourced in United States
SiGLO-Red is a fluorescent reagent used for the labeling and detection of proteins in Western blotting and other protein analysis techniques. It emits red fluorescence when excited by light of the appropriate wavelength.
Automatically generated - may contain errors
Lab products found in correlation
Nucblue, by Thermo Fisher Scientific (2 mentions)
Ix81 zdc microscope, by Olympus (2 mentions)
Growth factor reduced matrigel coated invasion chambers, by BD (2 mentions)
Ix81 zdc inverted epifluorescence microscope, by Olympus (2 mentions)
Amaxa cell line nucleofector kit 5, by Lonza (1 mentions)
Oligofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Siportneofx reagent, by Thermo Fisher Scientific (1 mentions)
Dharmafect sirna transfection reagent, by Horizon Discovery (1 mentions)
Rpmi 1640, by Sartorius (1 mentions)
9 protocols using siglo red
1
Efficient siRNA Knockdown in Cell Lines
2
Investigating Lamin A/C Knockdown in HeLa Cells
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HeLa cells were originally obtained from the American Type Culture Collection (ATCC) and were cultured as described previously [19 (link)]. The siRNA used in this study was Lamin A/C siRNA [20 (link)] obtained from Genesearch (Shanghai, China). siGlo red (Dharmacon, Lafayette, CO) was used as a qualitative transfection indicator as transfection efficiency.
3
In vivo siRNA and ETP Injection
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Example 15
For the in vivo injection of siRNA, a siRNA mixture specific to rat PrxII (200 nM) was premixed with a siPORT™ NeoFX™ reagent according to the instructions of the manufacturer (Ambion). Immediately after the balloon injury, the siRNA-injected mixture (200 μL) was injected through a catheter after briefly washed with Opti-MEM. After the incubation for 15 minutes, the blood flow was resumed. A siGLO-Red (Dharmacon), which is fluorescent dye-conjugated control siRNA, was used for optimizing in vivo injection efficiency. Similarly, the ETP compound (200 nM in DMSO) was injected through a catheter and incubated for 30 minutes.
4
In Vitro Invasion Assay Protocol
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In vitro invasion assays were performed as previously described59 (link). In brief, 1.5 × 105 cells were plated in the top wells of Growth Factor Reduced Matrigel-coated invasion chambers (8 µm pore size, BD Bio Coat). Media containing 5% serum was added to the lower chamber, and cells were allowed to invade along the serum gradient for 18 h at 37 °C. The assay was fixed with 3.7% PFA for 20 min and stained with NucBlue (Invitrogen) to visualize the nuclei. When siRNA-transfected cells were used, siGLO-Red (Dharmacon, Lafayette, CO, USA) was co-transfected in the cells to identify siRNA-treated cells. The membrane was detached from the chamber and mounted on a coverslip, and 10 random fields of view were imaged across the membrane at 20× magnification on an IX81-ZDC microscope (Olympus, Tokyo, Japan). The number of invading cells was counted manually with ImageJ software by thresholding onto the nucleus, and data are reported as the means of 3 experiments for each condition.
5
Rat-specific siRNA Delivery to Balloon-Injured Carotid Arteries
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The rat-specific siRNA SMART pools (Thermo Scientific Dharmacon, 200 nM) were premixed with siPORTTM NeoFX reagent following the manufacturer’s instructions (Ambion). The common carotid arteries were balloon-injured and briefly washed with Opti-MEM. The transfection pre-mix (200 μl) was then administered through the catheter with punctured balloon [19 (link), 25 (link)]. The vessel was incubated for 15 min for the efficient transfection and then ligated. A fluorescent dye-conjugated control siRNA (siGLO-Red, Dharmacon) was used for optimizing the intramural transfection efficiency.
6
Invadopodia Dynamics Imaged in MTLn3 Cells
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MTLn3 cells were transfected with cortactin-miRFP703 and EGFP-Tks5101 (link) before plating on gelatin-coated coverslips for 16 h. The cells were imaged every 2 min for 4 h on an IX81-ZDC inverted epifluorescence microscope at 60× magnification (Olympus). Invadopodia lifetimes were quantified manually for at least 30 invadopodia from at least 10 cells per condition in at least 3 experiments. Control and siRNA conditions were imaged on the same day for each experiment. Cells expressing siRNA and scrRNA were identified by co-transfection with siGLO-Red (Dharmacon).
7
Transfection of Pancreatic Islet Cells
8
Quantifying Invadopodia Dynamics in MTLn3 Cells
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MTLn3 cells were transfected with cortactin-miRFP703 and EGFP-Tks5 68 before plating on gelatin-coated coverslips for 16 h. The cells were imaged every 2 min for 4 h on an IX81-ZDC inverted epifluorescence microscope at 60× magnification (Olympus).
Invadopodia lifetimes were quantified manually for at least 30 invadopodia from at least 10 cells per condition in at least 3 experiments. Control and siRNA conditions were imaged on the same day for each experiment. Cells expressing siRNA and scrRNA were identified by co-transfection with siGLO-Red (Dharmacon).
Invadopodia lifetimes were quantified manually for at least 30 invadopodia from at least 10 cells per condition in at least 3 experiments. Control and siRNA conditions were imaged on the same day for each experiment. Cells expressing siRNA and scrRNA were identified by co-transfection with siGLO-Red (Dharmacon).
9
In Vitro Cell Invasion Assay
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In vitro invasion assays were performed as previously described 39 . In brief, 1.5 × 10 5 cells were plated in the top wells of Growth Factor Reduced Matrigel-coated invasion chambers (8 µm pore size, BD Bio Coat). Media containing 5% was added to the lower chamber, and cells were allowed to invade along the serum gradient for 18 h at 37°C.
The assay was fixed with 3.7% PFA for 20 min and stained with NucBlue (Invitrogen) to visualize the nuclei. When siRNA-transfected cells were used, siGLO-Red (Dharmacon, Lafayette, CO, USA) was co-transfected in the cells to identify siRNA-treated cells. The membrane was detached from the chamber and mounted on a coverslip, and 10 random fields of view were imaged across the membrane at 20× magnification on an IX81-ZDC microscope (Olympus, Tokyo, Japan). The number of invading cells was counted manually with ImageJ software by thresholding onto the nucleus, and data are reported as the means of 3 experiments for each condition.
The assay was fixed with 3.7% PFA for 20 min and stained with NucBlue (Invitrogen) to visualize the nuclei. When siRNA-transfected cells were used, siGLO-Red (Dharmacon, Lafayette, CO, USA) was co-transfected in the cells to identify siRNA-treated cells. The membrane was detached from the chamber and mounted on a coverslip, and 10 random fields of view were imaged across the membrane at 20× magnification on an IX81-ZDC microscope (Olympus, Tokyo, Japan). The number of invading cells was counted manually with ImageJ software by thresholding onto the nucleus, and data are reported as the means of 3 experiments for each condition.
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