Cell viability assays following treatment with aDT–eIF-3b

1)    Prepare cell culture

a.     Coat 96-well plates with Poly-L-ornithine (0.01%) for 30 minutes at 37 ^oC. Remove PLO solution and replace with laminin solution (5 mg/mL) overnight at 37 ^oC.

b.     Wash wells once with PBS.

c.     Passage cells and prepare cell stock solution in their culture medium at a concentration of 8000 cells/75mL (cell density should be determined empirically for different cell types). Plate 75mL cell solution per well.

2)    Prepare treatment

a.     Prepare a concentrated aDT-siRNA solution at least 4x the concentration of your highest desired treatment concentration in Opti-MEM.

b.     Dilute aDT-siRNA to 4x the desired treatment concentration with Opti-MEM and plate 25mL per well.

c.     For negative control, add 25mL Opti-MEM per well.

3)    24h after plating, add 50mL cell culture media per well.

4)    Viability reading

a.     48h after plating, remove all media in well, and replace with 90uL media.

b.     Add 10mL PrestoBlue viability reagent

c.     Incubate plate at 37 ^oC for 2h.

d.     Read the PrestoBlue signal on a fluorescence plate reader (Em./Ex. 535-560⁄590-615).

e.     Quantify cell viability as a percentage of untreated controls.

Category