Standard Protocols for Vitamin E And

STANDARD PROTOCOLS FOR VITAMIN E AND

VEHICLE TREATMENTS OF CELLS

KLINE LAB 2/96

VITAMIN E SUCCINATE (d-a- tocopherol succinate; Sigma #T-3126)

Weigh out 10 mg of Vitamin E succinate, dissolve in 1 ml of absolute Ethanol (heating in 370C water bath will help vitamin E go into solution). This is your STOCK solution.

Then proceed with the following dilution scheme:

1:100 (MEDIA) 1:10 (MEDIA)

10mg/ml à100 mg/ml à10 mg/ml à etc.

(100%EtOH) 1% EtOH 0.1%EtOH

(for each of the additional dilutions use media containing 0.1% EtOH, this same dilution scheme can be done starting with 100% DMSO)

VEHICLE (succinic acid; Sigma #S-2378 and EtOH equivalents)

Approximately 1/5(20%) of vitamin E Succinate (d-a-tocopherol acid succinate; Sigma #T-3126) is succinate. i.e.: in 10 mg/ml vitamin E succinate there is 2 mg/ml succinate.

Approximately 43% of the Succinic Acid (disodium hexahydrate; Sigma #S-2378) is succinate. For convenience, round to 50% (This means that only 1/2 of the amount you weigh out is succinate). Therefore, if you need a 2 mg/ml succinate equivalent , you would use 2 x (2 mg)/ml, which would be 4 mg/ml of Succinic Acid (Sigma #S-2378).

To make this a true vehicle control you need to add the equivalent F.C. of EtOH which you used in your Vitamin E solution (i.e. from above 0.1%). Succinic Acid is insoluble in Ethanol therefore it must be dissolved in media before the Ethanol is added.

An equivalent Vehicle for the 10 mg/ml Vitamin E succinic acid treatment would be:

Weigh out 20 mg Succinic Acid, dissolve in 5 ml media, then add 5 ml of EtOH, then proceed with one of the following dilution schemes:

1:50 (MEDIA) 1:10 (MEDIA)

2mg/ml à40mg/m1 à 4mg/ml

(50% Media/50% EtOH) (1% EtOH) (0.1% EtOH)

Other Forms of Vitamin E

dl-a-Tocopherol (Sigma # T-3251) and d-a-Tocopherol (Sigma # T-3634)

Bring these forms to room temperature and then dissolve 0.105 ml of the appropriate form in 0.895 ml of 100% DMSO. This will give you a 100mg/ml stock. Warm in 370C bath then proceed with the following dilution scheme:

1:10 (pre-warmed FCS) 1:5 (MEDIA) 1:20 (MEDIA)

100mg/ml à 10mg/ml* à2mg/ml à 0.1mg/ml

100% DMSO 10% DMSO 2% DMSO 0.1% DMSO

* Heat this dilution for 1 hour at 370C

DMSO Control

Spike the appropriate media with either 0.5 or 0.1 % Absolute Ethanol or DMSO(percentage should match the highest percentage in your treatment groups.)

Vitamin E Acetate (Sigma; #T-33760) MW. 472.8. 100 mg/ml in EtOH stock solution.

Vihicle (Acetate + EtOH) Sodium Acetate Baker #3462-01) MW. 136.8. 100 mg/ml of vitamin E Acetate = 9 mg/ml of acetate. Sodium Acetate have about 50% acetate, therefore, 18 mg/ml of sodium acetate = 9 mg/ml of Acetate.