DNA analysis of stimulated lymphocytes by automatic sampling for flow cytometry

Abstract

A microsample delivery system (MSDS) was tested for automatic flow cytometry (FCM) analysis of DNA synthesis in stimulated human peripheral blood lymphocytes (PBL) cultivated in wells of microtiter plates. After incubation, either for 1-3 days with phytohemagglutinin, concanavalin A, and pokeweed mitogen, or for 7 days with allogenic PBL, the cells, while in the wells, were washed in hypotonic Tris buffer and stained with ethidium bromide-RNAse solution.The results obtained from quintuplicate replicated wells, each of the five containing the same control or stimulated cultures, were reproducible in terms of the number of nuclei counted in each histogram of control, mitogen-stimulated PBL, and mixed lymphocyte cultures (MLC). Using a computer program that superimposes histograms and calculates their differences on the scale of fluorescence intensity, it was possible to quantify the intensity of the response to the mitogenic stimuli.This approach to the study of lymphocyte proliferation offers not only a simpler and faster analysis of DNA synthesis than the method of 3H-thymidine incorporation, but it also allows for the analysis of other FCM parameters, such as forward and 90" light scatter and double fluorescence labelling of PBL nuclei.

Sequential Assessment of Cell Cycle S Phase in Flow Cytometry: A Non‐Isotopic Method to Measure Lymphocyte Activation In Vitro

“…As could have been expected, owing to the polyclonal stimulation it generates, PHA activation yielded the maximal signal observed on day 3, with highly significant numbers of S and G2+M cells at this time point. This is consistent with data from isotopic techniques indicating that 72 hrs is the optimal incubation time to demonstrate PHA stimulation [2,10]. Little is known of the expression of mitogen receptors by newly generated daughter lymphocytes, but our data suggest that there is no further stimulation of these cells by the remaining PHA, or that all has been bound to responding lymphocytes.…”

Gingipains in the culture supernatant of Porphyromonas gingivalis cleave CD4 and CD8 on human T cells

“…PBMC (1 × 10 6 cells/ml) were cultured with PHA (6 µg/ml) for 3 days or Con A (20 µg/ml) for 4 days at 37°C in a humidified atmosphere containing 5% CO 2 . After incubation, the analysis of DNA synthesis was performed according to the procedure reported previously (12). Results were expressed as the Stimulation Index that was calculated on the basis of fluorescence intensity of nucleus gated in the fluorescence channels including the S + G 2 /M regions.…”

Identification of Antigen-Specific Hormone-Resistant Blood Lymphocytes to Predict Efficiency of Glucocorticoid Therapy in Bronchial Asthma

“…АГ/К7 А + П/АГ 0,38 0,39 Возможность использования проточной ДНК-цитометрии для оценки митогенстимулированной пролиферации как альтернативы традиционного радиоактивного теста была обоснована рядом работ зарубежных исследователей [7,11,19]. Результаты исследований свидетельствуют о том, что ввиду высокой воспроизводимости, точности, чувствительности, скорости анализа, безопасности и относительной простоты оценка пролиферации лимфоцитов методом ДНК-цитометрии может быть более предпочтительной для лабораторной практики по сравнению с тестом, где используется радиоактивная метка.…”