ON-TARGETplus SMARTpool siRNA constructs for human ARP3 and siGLO transfection indicator (Dharmacon) were transfected as per manufacturer’s protocol. Knockdown cells selected for analysis had at least four times the background levels of siGLO red.
Siglo transfection indicator
Manufactured by Horizon Discovery
The SiGLO transfection indicator is a fluorescent reporter system designed to monitor transfection efficiency in cell-based experiments. It provides a visual representation of the success of the transfection process.
Automatically generated - may contain errors
3 protocols using siglo transfection indicator
1
Knockdown of Human ARP3 with siRNA
2
ASPP2κ siRNA Lipofection Protocol
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Specific ASPP2κ siRNA constructs were developed to be used with standard lipofection transfection techniques following the manufacturer protocols.
The constructs or a scrambled siRNA control were lipofected into leukemia cell lines or freshly harvested native leukemia cells using a lipofectamine 2000®-based protocol (Invitrogen, CA). Briefly, 5 × 105 cells/well were plated in a 24-well plate and cultured in medium containing serum without antibiotics. Constructs are diluted in serum-free medium and mixed with a lipofectamine. The mix is then added to the cell suspension followed by incubation at 37 °C. Transfection efficacy was independently validated by siGLO transfection indicator (Dharmacon).
The constructs or a scrambled siRNA control were lipofected into leukemia cell lines or freshly harvested native leukemia cells using a lipofectamine 2000®-based protocol (Invitrogen, CA). Briefly, 5 × 105 cells/well were plated in a 24-well plate and cultured in medium containing serum without antibiotics. Constructs are diluted in serum-free medium and mixed with a lipofectamine. The mix is then added to the cell suspension followed by incubation at 37 °C. Transfection efficacy was independently validated by siGLO transfection indicator (Dharmacon).
3
Silencing MMP-9 in Huh7.5 cells
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Huh7.5 (106) cells were seeded and reverse transfected in 6 well plates, in DMEM-E2 minus for 24 hr, with 20 nM final concentration of small interfering RNA (siRNA) following the manufacturer protocols (Dharmacon, Inc., Lafayette, CO). siRNA, Dharmafect 4 Transfection reagent, 5X siRNA buffer and siGLO Transfection Indicator were obtained from Dharmacon. siRNA specific to MMP-9 (ON-TARGET plus Human MMP-9 siRNA “SMART pool”, Target sequences: GCAUAAGGACGACGUGAAU, GGACCAAGGAUACAGUUUG, GCGCUCAUGUACCCUAUGU, and GAACCAAUCUCACCGACAG). MMP-9 sequence-Scrambled siRNA was used as a negative control (ON-TARGET plus Non-Targeting siRNA, Target sequence: UGGUUUACAUGUUUUCCUA).
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