Scientific research protocol
Scientific studies done with Basquade
Work protocol
Cinetics tests of cell proliferation
These tests will be done on 2 cell types:
1) Tumoral cells: tumoral lymphoid line of murine origin BW 5147
that expresses the haplotype H-2k, CD3+ and the TCRab, whci are
tested routinarily by flux citometry using specific antibodies
against the correspondant surface markers.
2) T Lymphocites normals coming from mice lymph glands of the
variety BALG/c 3-4 months old, obtained in an ascetic way.
In vitro tests will be done in sterile condition in 96 wells culture
microplates in a final volume of 0,2 ml. The effect on the cell
proliferation of the mix to test will be evaluated at different
culture times (24, 48, 72 and 96 hours) and in absence (Control)
or presence of different concentrations of mix to determine the
cynetic action of the product.
The concentrations to test will be dilutions in series to the
half in a range of concentrations that includes the proposed dose
to use in humans (from 1/250 to 1/4000). The cell proliferation
will be evaluated through a technique of incorporation of Timidna
tritiada (20 Ci/mnol). For that purpose, the cell cultures will
be pulsed for a 16 hour period in the case of normal lymphocites
and 6 hours for tumoral cells with Timidina tritiada, after what
the cells nucleus are retained by filtration of glass fibre paper,
Whatmann GF/A. The radioactive Timidine incorporated to the DNAwill
be quantified in a Wallac liquid twinkle counter.
The cells concentrations to use will be: for the tumoral line
of 3 per 105 cells/ml (optimum final cell concentration) and for
the normal cells of 2 per 106 cells/ml. Both cell types will be
cultivated in RPMI 1640 suplemented with 10% fetal bovine serum
and 2mM of glutamine and in presence of the peniciline antibiotics
(100 U/ml) and streptomicine (100 ug/ml).
