Experiment 1: Effects of H2O2 on viability of SHSY5Y neuronal cells determined by Trypan

Blue dye exclusion.

(a)

24-well plates were seeded with 5 x 105

cells/well and incubated overnight at

37C.

(b)

A fresh stock of 1 mM H2O2 was prepared in serum free media. A series of five

dilutions in serum free medium to determine the effect of the H2O2 on the cells

were prepared.

(c)

Dilutions of the stock H2O2.stock (30 mM) in serum free medium were prepared.

(d)

A 24 well plate with SHSY5Y neurons already seeded onto it was treated with

500ul of each H2O2.concentration along with a medium alone control

concentrations were tested in quadruplicate.

(e)

The plate was incubated for 1 h at 37C.

(f)

Following incubation media was removed from each well, and 100 l of 4%

Trypan Blue was added to each well.

(g)

Plates were then photographed and the images are displayed on mybeckett for

you to analyse. You can count cells that have excluded Trypan Blue (viable cells)

plus cells that have taken up the blue dye (dead cells). The ratio of live cells/total

cells counted can be used to calculate the percentage viability under each

condition