ABSTRACT
One method for examining cell cycle kinetics by flow cytometry uses continuous DNA labeling with bromodeoxyuridine (BrdU), a thymidine analogue. Upon incorporation into DNA, BrdU causes stoichiometric quenching of the DNA fluorochrome Hoechst 33258. After counterstaining with a secondary DNA fluorochrome (e.g., ethidium bromide), the analyst can distinguish cells in different phases of the cell cycle over a number of mitotic cycles with flow cytometry. In this report, we describe a modification of the flow cytometric BrdU-Hoechst assay that allows combined analysis of cell proliferation and immunophenotyping at the single cell level. To demonstrate an application of this method, human peripheral blood mononuclear cells were stimulated with tetanus toxoid or interleukin-2 for up to 6 days in the presence of BrdU, harvested, and immunostained for the cell surface markers CD3, CD4, CD8, CD14, CD19, and the cytokine receptor, CCR5. We used four-color flow cytometry analyses to simultaneously measure cell proliferation and surface marker expression, for the purpose of immunophenotyping and identifying specific cell subsets responding to antigen stimulation. Our successful application of this method suggests that it may be used to study immune responses at the molecular and cellular level and to identify mechanisms of immune system modulation.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/analysis , Antigens, Differentiation, T-Lymphocyte/analysis , Immunophenotyping/methods , Lymphocyte Subsets/classification , Bisbenzimidazole/chemistry , Bromodeoxyuridine/chemistry , Cell Cycle , Cell Membrane Permeability , Cells, Cultured , DNA/analysis , Dactinomycin/analogs & derivatives , Dactinomycin/chemistry , Ethidium/chemistry , Fixatives/chemistry , Flow Cytometry , Fluorescent Dyes/chemistry , Formaldehyde/chemistry , Humans , Lymphocyte Activation , Lymphocyte Subsets/chemistry , Lymphocyte Subsets/cytology , Polymers/chemistryABSTRACT
It is unclear whether both bone resorption and formation are affected by glycemic control, and contribute to diabetic osteopenia. In this study, 20 patients with noninsulin-dependent diabetes mellitus (12 men and 8 postmenopausal women) and 20 healthy control subjects (10 men and 10 postmenopausal women) were examined at baseline and 2 months. The diabetic patients showed an improvement of glycemic control (decreased HbA1c) at the second measurement. Analysis of variance showed that there was no effect of gender on the variables that increased with improved glycemic control, and therefore results are presented for both male and female subjects. Baseline values of serum osteocalcin, a marker of formation, were significantly lower in diabetic patients compared with healthy subjects (2.5 +/- 1.3 versus 4.4 +/- 1.4 ng/ml; P = 0.0006), but markers of bone resorption [urinary pyridinoline (PYD), deoxypyridinoline (DPD)] did not differ. Improved glycemic control in diabetic patients resulted in increased values of PYD (P = 0.012), DPD (P = 0.049), serum osteocalcin (P = 0.001), and serum insulin-like growth factor I (IGF-I, P = 0.003), but no change in serum parathyroid hormone or 25-hydroxyvitamin D. In diabetic patients there were inverse correlations for the percent change from baseline to improved glycemic control for osteocalcin and HbA1c (r = -0.53; P = 0.016) and glucose (r = -0.46; P = 0.050). These data suggest that improved glycemic control is accompanied by an increase in bone turnover for male and female diabetic patients, possibly mediated by increased levels of circulating IGF-I.