Stivven's List: Methods in mol bio

Caspase assay

150 uM, 6 mL

150 nmol/mL --> 900 nmol, so about 1umol

DEVD mr= 675

€211/5 mg ---0.0074 mmol in total, about 7.4 umol, enough for about 8 plates wonder how economical that is??? 

http://www.biotechniques.com/multimedia/archive/00011/03345dd02_11120a.pdf very useful . . . .

TTTNotes: it is significantly more economical to create the assay in-house, as the cost works out at about 20c per 25mL of working reagent, compared with €170 for 25 mL of the prepared reagent (both from Sigma). A paper from 2000 outlined the concentration of resazurin in the commerical alamar blue reagent at 440 micromol/L

According to the Eur. J. Biochem. 267, 5421±5426 (2000) FEBS 2000 the final conc of Resazurin in the AB ragent is 440 uM (Mr: 251.17). This is used at a concentration of 10% final volume, so 10 uL in a 96 well plate.

251.17 ug/L = 1 uM --> 110.52 mg/L =440 uM

For 25 mL solution, should aliquot 2.77 mg 

Taking 3 mg and adding to 27.15 mL gives a working solution which would be sufficient for about 25 x 96 well plates

1 g = €72, so 3mg = .072*3= €0.216 per 25 mL, rather than €170 form Sigma, approx 1c/plate

NB Establish time course of exposure to the detection reagent for each cell line to be used - 2 hr prob OK. Also not that fluorescent resorufin can be broken down at long time periods reducing fluor...also needs to make sure that the cell desnsity is also checked (cf http://humrep.oxfordjournals.org/content/22/5/1304.full)

Resazurin Reagent:

3 mg of reagent is dissolved in 27.15 mL of balanced salt solution (? HBSS or PBS)

Assay protocol:

1) Treat cells with chemical of interest for appropriate interval (24, 48, 72 hours)

2) Remove medium & replace with phenol red free medium containing 10% resazurin reagent

3) Incubate cells for 2 hours 

4) Assay decrease in colour (at 600nm with ref of 690nm) and/or increase in fluorescence

Controls:

Empty wells; untreated wells; ??? Positive control

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

Intracellular ROS

D6883-50MG

; €51.10

MTT Assay

5mg/mL using only 10 uL, so  that means 50ug per well of a 96 well plate....about 1€ per plate

100 mg is €22.50 from sigma (M2128 SIGMA, Thiazolyl Blue Tetrazolium Bromide
incubate for detection at 0.5mg/mL....need 100 uL per well, so adding the 50 ug per well....

Method:
1. Add 10 uL of a 5 mg/mL solution of MTT per well.

1. LDH Assay works out at 2.8 c/test so economic and easy to work with

2. Resazurin works out at about 1c/plate

LDH Assay

Based on protocol form http://www.opsdiagnostics.com/applications/applicationtable/Lactate%20Dehydrogenase%20Protocol.htm

*TRIS base: 100g for 50:10-> €11.12 per L; 47:80 per 100g-->€5.07 per L for a total of €16.20

*Phenazine Methosulfate (Sigma P-9625), i.e., PMS, by dissolving 0.9 mg into 100 ul water (32:30 per 1000mg) 

 = 0.029 cost; works out at 3c/100uL

*Dissolve INT (Sigma I-8377 or equivalent) into DMSO (Sigma D-8779 or equivalent) at a concentration of 3.3 mg/100 ul DMSO, (€31:90 per 250 mg)

=0.42 cost; works out at 42c/100uL

*Prepare NAD (Sigma N-0632 or equivalent) by adding 8.6 mg NAD to 2.3 ml water. - €44:80 per 1000mg

=0.385 cost; works out at 38.5c/2.3mL

*Prepare by adding 49 mg lithium lactate (Sigma L-1500 or equivalent) into 2.5 ml of water. -(€26:50 per 5000mg)

=0.52 cost; 52c for substrate

*Mix 100 ul PMS, 100 ul INT, and 2.3 ml NAD solution.,Total 2500uL and 50 uL per assay

Use triton X 100 at 1% for 100% lysis of the cells.....

Mix 3c PMS; 42 c INT; 38.5 c NAD = total of 83.5c in total for 2.5 mL=>1.67 c/50 uL

Substrate= 52c total for 2.5 mL                                                        =>1.04 c/50 uL

Buffer=                                                                                          =>0.08 c/50 uL

Total for the assay                                                                          => 2.79 c/150 uL reagent

In total the LDH assay would cost 2.8 c