METHOD OF QUANTITATIVE ESTIMATION OF APOPTOSIS MODIFIED BY ORGANIC LOW-MOLECULAR COMPOUNDS Russian patent published in 2012 - IPC G01N33/49 

FIELD: medicine.

SUBSTANCE: invention described a method of quantitative estimation of apoptosis modified by organic low-molecular compounds, involving patient's blood sampling, recovering mononuclear cells from the sample and cell incubation with the analysed compound wherein the mononuclear cells are recovered from the sample by introducing ethylene diamine tetraacetate into the sample; the sample is diluted by aqueous saline, layering on ficoll-verografin 3 ml at density gradient 1.077 g/ml, then centrifuged for 30 min at 1500 rpm; the mononuclear cells are sampled; then the recovered cells are washed in saline; said cells are resuspended in an aqueous working staining buffer IX Annexin V Binding Buffer to the concentration of 1×10⁶ cells/ml. The working staining buffer IX Annexin V Binding Buffer represents mixed concentrated staining buffer 10X Annexin V Binding Buffer with distilled water in volume ratio 1:9 respectively; then the prepared cells are incubated with the analysed organic low-molecular compound to produce a test sample; the prepared cells are incubated without said compound added to produce a reference sample; the prepared cells are incubated with the analysed organic low-molecular compound and without it for 1 hour at temperature 37°C; the incubation is followed by introducing Annexin V and propidium iodide into the test and control samples; the samples are re-incubated in the dark at room temperature for 15 min; the working staining buffer IX Annexin V Binding Buffer is added that is followed by cytometric analysis of the prepared samples to determine the Annexin V content; the numerical Annexin V contents in the control and test samples are compared, and the relation of the greater Annexin V content to the smaller one is used to estimate apoptosis modified by the organic low-molecular compound with said relation being 1.5 and more enables stating apoptosis - a critical condition of the programmed cell death caused by action of said organic compound.

EFFECT: method provides higher accuracy of quantitative estimation of apoptosis modified by the organic low-molecular compounds with enabled further estimation of immune deficient conditions in population.

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