Stivven's List: Sterol Biochemistry

Overall Cholestanol Detox Pathway Plan

Hypothesis: if formation of cholestanol is a detoxificaiton pathway, then there will be a decreasing toxicity with more mature intermediates of the cholestanol formation pathway from C4

Specific Objectives:

1) Determine the effect of treatment of cell lines (e.g. HepG2, Hep3B, Neuroblastoma) with 8 different steroids (5nMol-10uM) on LDH release

2) Determine the effect of treatment of cell lines (e.g. HepG2, Hep3B, Neuroblastoma) with 8 different steroids on resazurin reduction

3) Determine the effect of treatment of cell lines (e.g. HepG2, Hep3B, Neuroblastoma) with 8 different steroids on chromatin condensation

4) Determine the effects of . . . 

8 different sterols this is how to change it

Stock solution of sterols at 1mmol/L & 0.1 mmol/L & 0.01 mmol/L

96 well culture is 100-200 uL per well, want max 0.5% EtOH and also add CD in final solution (?? concentration)

- want to do concentrations at: 0.00, 0.005, 0.01, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10.0 micromol/L

- 11x concentrations including vehicle; leaves one row of cells available for non-treated 

- do 8x replicates, prep sufficient for 10 wells (i.e. 1 mL of reagent at 100 uL/well)

10 uM => 1:100 dilution of the 1mM stock, so 10 uL of stock + 0 uL EtOH per mL of complete media

5 uM => 5 uL stock + 5 uL EtOH per mL complete medium

2.5 uM => 2.5 uL stock + 7.5 uL EtOH per mL complete medium

1.0 uM => 1 uL of stock + 9 uL EtOH per mL of complete media 

0.50 uM => 5 uL low-stock + 5 uL EtOH per mL complete medium

0.25 uM => 2.5 uL low-stock + 7.5 uL EtOH per mL complete medium

0.10 uM => 1 uL low-stock + 9 uL EtOH

0.050 uM => 5 uL very low stock

0.025 uM => 2.5 uL very low stock

0.010 uM => 1 uL very low stock

0.005 uM => 0.5 uL very low stock