Determination of cellular thiol levels in individual viable lymphocytes by means of fluorescence intensity and polarization

• Published in: Journal of immunological methods Vol. 229; no. 1; pp. 23 - 34
• Main Authors: Zurgil, Naomi, Kaufman, Menachem, Solodiev, Inna, Deutsch, Mordechai
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 29-10-1999; Elsevier
• Subjects: Analysis of the immune response. Humoral and cellular immunity; Biological and medical sciences; Cellscan; Ethylmaleimide - pharmacology; Fluoresceins; Fluorescence Polarization; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Glutathione; Humans; Immunobiology; Jurkat Cells; Lymphocytes - chemistry; Organs and cells involved in the immune response; Phytohemagglutinins - pharmacology; Sulfhydryl Compounds - analysis; Thiols; Fluorescence polarization; FI, fluorescence intensity; FDA, fluorescein diacetate; CMF, chloromethyl fluorescein; Thiols; GSH, glutathione; CMFDA, chloromethyl fluorescein diacetate; PBMC, peripheral blood mononuclear cells; Cellscan; PHA, phytohaemagglutinin; LP, low polarization; GST, glutathione S transferase; FPR, fluorescence polarization ratio; PBS, phosphate-buffered saline; COM, center of mass; GSSG, glutathione disulfide; DMSO, dimethyl sulfoxide; HP, high polarization; NEM, N-ethyl maleimide; FP, fluorescence polarization; Glutathione; Human; Polarization; Thiol; Mononuclear cell; Fluorescent labelling; Quantization; Cellular immunity; Method; In vitro; Lymphocyte; Quantitative analysis
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(99)00112-X

Cellular thiol levels regulate lymphocyte proliferation and death and play a significant role in the immune response. Therefore, the ability to analyze the total protein and non-protein thiol compounds and their distribution among individual living lymphocytes is of great importance. A quantitative measurement of intracellular sulphydryl groups in living lymphocytes using the Cellscan mark
F (CS-
F) cytometer, in conjunction with the probe CMFDA, is described. This technique permits the detection, identification, and study of sub-populations and single cells in a sample of heterogeneous lymphocytes. The Cellscan apparatus is a laser based scanning cytometer incorporating a unique cell carrier which allows repeated, high-precision measurements of fluorescence intensity (FI) and fluorescence polarization (FP) to be made on intact individual living cells under controlled physiological conditions. The discernible effect of fluorophore molecules bound to thiols having a higher FP than free molecules was used to estimate their relative fractions in living lymphocytes. The results were more conspicuous when the ratio between FP measured at two wavelengths (FPR) of the fluorogenic molecules was used for analysis. In addition, the intracellular dynamic changes in the FI, FP and FPR of the fluorescent probe were also monitored. The cellular sulphydryl content of each lymphocyte within a population was recorded by the CS-
F, and sub-populations or individual cells were classified according to their thiol levels and their metabolic rates. Changes in thiol concentration were observed following mitogenic activation of peripheral lymphocytes.