Investigation Study's Protocol

Scientific studies made with GREEN SAP
WORK'S PROTOCOL

Kinetic essays of cellular proliferation
These essays will be made on 2 cellular types:

1. Tumor cells: lymphoid tumor line of murin origin BW 5147 which express H-2k haplotype, CD3+ and TCRaß, the ones are tested usually by flux citometry using specific antibodies against the correspondent surface markers.

2. Normal Lymphocytes T from lymphatic nodes of rats from BALG/c with 3-4 months of age, obtained in a aseptic way.
The essays in vitro will be done in sterility conditions in cultivations of 96 wells in a final volume of 0.2 ml.

The effect on cellular proliferation of the mixture to be essay will be evaluated at different times of cultivation (24, 48, 72 y 96 hs.) and in absence (Control) or presence of different concentrations of mixture so as to determine the kinetic action of the product. The concentrations to be tried will be series of dilutions in half in a rate of concentrations that include the proposed dose to be used in human beings (from 1/250 till 1/4000).

Cellular proliferation will be evaluated with the trytiade tymidin incorporation technical (20 Ci/mmol). For this, the cellular cultivations will be pulsed for a period of 16 hs in the case of normal lymphocytes and of 6 hs for tumor cells with trytiade tymidin, after this cellular nucleus are kept by glass fiber paper filtration, Whatmann GF/A. The radioactive tymidin incorporated to DNA will be counted in a Wallac count of liquid glitter.

The cells concentration to be used will be: for tumor line of 3x105 cells/ml (final optimum cellular concentration) and for normal cells 2x106 cells/ml. Both types will be cultivated in RPMI 1640 supplemented with 10% of bovine fetal plasma and 2mM of glutamine and in presence of penicillin (100 U/ml) and streptomycin (100 µg/ml).