esiRNA human PTPN3 (esiRNA1)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add diluted siRNA to diluted Lipofectamine™ 3000 Reagent(1:1 ratio). Incubate for 10-15 minutes at room temperature and add to cells. Incubate cells for 3 days at 37°C.

Protocol tips
Add diluted siRNA to diluted Lipofectamine™ 3000 Reagent(1:1 ratio). Incubate for 10-15 minutes at room temperature and add to cells. Incubate cells for 3 days at 37°C.

Publication protocol

The A2780CIS and A2780ADR cells were seeded in the six well plates and transfected
with esiRNA for 72 h. Cells were collected by tripsinization and washed twice in PBS, fixed in
70% chilled ethanol by adding dropwise into the samples while vortexing and stored at 4 °C until
analyzed. Immediately before analysis, cells were washed twice in PBS, and incubated with
250 μg/ml RNase A (Invitrogen) for 30 min at room temperature, followed by staining with PI
(Sigma) at 20 μg/ml final concentration for 30 min incubation in dark. The cell cycle distribution
and percentage of apoptotic cells were analysed using a FACS analyser (BD LSRFortessa). Ten
thousand cells were analysed for each sample. Appropriate gating was used to select the singlecell population. The percentage of cells in sub-2n phase was determined using software (BD
FACSDiva v6.0)

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for esiRNA human PTPN3 (esiRNA1) below.

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