ON-TARGETplus Human SLC7A2 (6542) siRNA - SMARTpool

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 5 X 10^5 cells per well in 6-well for for 20 h at 37C.	siRNA concentration-100 nM Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 5 min at RT and add to cells. Incubate cells for 6h at 37C and replace medium to incubate another 24-48 h.

Upstream tips
Seed 5 X 10^5 cells per well in 6-well for for 20 h at 37C.
Protocol tips
siRNA concentration-100 nM Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 5 min at RT and add to cells. Incubate cells for 6h at 37C and replace medium to incubate another 24-48 h.

Publication protocol

YAMC cells were maintained under permissive growth conditions in DMEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 U/ml IFN-γ in a humidified incubator with 5% CO2 at 33°C [24, 43]. These cells were plated in 96-well plates at 33°C for 20 h and hexadimethrine bromide (8 μg/ml) was added to the cells to enhance transduction efficiency. Lentiviral particles containing Slc7a2 or control shRNAs (Sigma) were added at an MOI of 5 and incubated for an additional 20 h at 33°C. Transduced cells were selected in complete medium with 10 μg/ml puromycin, which was replaced every 3–4 days for 3 weeks [24]. YAMC cells, transduced or not, were then transferred at 37°C in IFN-γ-free medium for 24 h before infection with C. rodentium.

Caco-2 and HEK 293T cells were grown in DMEM containing 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. These cells were plated at 5 X 105 cells per well in 6-well plates at 37°C for 20 h. Medium was changed to Opti-MEM I Reduced-Serum Media (Invitrogen) and cells were transfected using Lipofectamine 2000 (Life Technologies) with i) 100 nM ON-TARGETplus siRNAs (Dharmacon) directed against human SLC7A2 or scrambled siRNAs, or ii) 1 μg of the pLX304 plasmid vector expressing or not the human SLC7A2 gene. After 6 h, cells were washed, maintained 24–48 h in fresh medium, and then infected with C. rodentium or EPEC, in the presence or absence of 40 mM L-Lys.

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Manufacturer protocol

Download the product protocol from Dharmacon for ON-TARGETplus Human SLC7A2 (6542) siRNA - SMARTpool below.

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