Wiley

Bài tổng quan này mô tả nhiều phương pháp để nhận diện và phân biệt giữa hai cơ chế chết tế bào khác nhau, apoptosis và hoại tử. Đa phần các phương pháp này đã được áp dụng trong các nghiên cứu về apoptosis trong dòng tế bào bạch cầu HL-60 của người bị kích hoạt bởi các chất ức chế DNA topoizomeras I hoặc II, và trong các tế bào tuyến ức của chuột bởi cả chất ức chế topoizomeras hoặc prednisolone. Trong hầu hết các trường hợp, apoptosis chọn lọc đối với tế bào trong pha nhất định của chu kỳ tế bào: chỉ tế bào HL-60 pha S và tế bào tuyến ức G₀ bị ảnh hưởng chính. Hoại tử được kích hoạt bởi nồng độ quá cao của những loại thuốc này. Các đặc điểm tế bào sau đây đã được xác định có ích trong việc nhận diện kiểu chết tế bào: (a) Sự kích hoạt endonuclease trong tế bào apoptosis dẫn đến việc chiết xuất DNA có trọng lượng phân tử thấp sau khi tế bào bị thẩm thấu, dẫn đến giảm khả năng nhuộm bằng các fluoroquinone đặc hiệu với DNA. Đo hàm lượng DNA giúp nhận diện tế bào apoptosis và phát hiện được pha đặc hiệu của chu kỳ tế bào liên quan đến tiến trình apoptosis. (b) Tính toàn vẹn màng tế bào, mất trong tế bào hoại tử nhưng không mất trong tế bào apoptosis, đã được thăm dò bằng cách loại trừ iodua propidium (PI). Sự kết hợp giữa PI và Hoechst 33342 tỏ ra là một đầu dò tuyệt vời để phân biệt các tế bào sống, hoại tử, apoptosis sớm và muộn. (c) Điện thế xuyên màng ty thể, đo thông qua khả năng giữ rhodamine 123 được giữ nguyên trong tế bào apoptosis nhưng không trong tế bào hoại tử. (d) Bơm proton lysosome phụ thuộc vào ATP, thử nghiệm thông qua khả năng hút acridine orange (AO) trong môi trường sống, cũng được giữ nguyên trong tế bào apoptosis nhưng không trong tế bào hoại tử. (e) Phân tích bivariate của tế bào được nhuộm DNA và protein tiết lộ giảm đáng kể hàm lượng protein trong tế bào apoptosis, có lẽ do sự kích hoạt của protease nội sinh. Tế bào hoại tử, có màng bị rò, có hàm lượng protein tối thiểu. (f) Nhuộm RNA cho phép phân biệt giữa tế bào G₀ và G₁ và như vậy có thể chứng minh rằng apoptosis lựa chọn tế bào tuyến ức G₀. (g) Sự giảm trong tán xạ ánh sáng phía trước, được đi kèm bởi hoặc không có thay đổi (tế bào HL-60) hoặc tăng (tế bào tuyến ức) của tán xạ góc phải, là những thay đổi sớm trong apoptosis. (h) Độ nhạy của DNA in situ đối với sự suy thoái, tăng trong tế bào apoptosis và tế bào hoại tử. Đặc điểm này, được thăm dò bằng cách nhuộm với AO ở pH thấp, cung cấp một thử nghiệm nhạy cảm và sớm để phân biệt giữa các tế bào sống, tế bào apoptosis và tế bào hoại tử, cũng như để đánh giá đặc điểm pha chu kỳ tế bào của các tiến trình này. (i) Phương pháp chuyển dịch nick in situ sử dụng triphospohonucloside gắn nhãn có thể được sử dụng để tiết lộ đứt gãy sợi DNA, để phát hiện giai đoạn rất sớm của apoptosis. Dữ liệu cho thấy rằng lưu lượng tế bào học có thể được áp dụng trong nghiên cứu cơ bản về cơ chế sinh hóa và phân tử của apoptosis, cũng như trong lâm sàng nơi khả năng theo dõi các dấu hiệu sớm của apoptosis trong các mẫu từ các khối u của bệnh nhân có thể dự đoán kết quả của một số phác đồ điều trị. © 1992 Wiley-Liss, Inc.

A new modification of our detergent technique for the preparation of nuclei for flow cytometric DNA analysis is described. The attainment of low coefficients of variation of the peaks and of quantitative staining of nuclei from different tissues was a problem with the original method. This was solved in the new modification by trypsinization of the unfixed nuclei. The nuclei were stabilized by spermine. A simple procedure for long‐term storage of samples at —80°C was integrated into the method. The fluorescence of the nuclei was stable for at least 3 hours after staining. Light exposure protection of the samples was essential. No cell loss was caused by storage or staining. The method was successfully applied on samples including: (a) Normal tissues— human lymphocytes, granulocytes and spleen. Mouse lymphocytes, bone marrow, spleen, liver, kidney and thymus. (b) Human neoplasms— lung cancer, breast cancer, lymphoma, leukemia, bladder cancer and cancer of the oral cavity. (c) Human tumors in nude mice— breast cancer, lung cancer, melanoma and colon cancer. (d) Mouse ascites tumors— JB‐1, L 1210, Ehrlich and P 383. It therefore seems well suited as a routine clinical procedure.

Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non‐vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7‐amino‐actinomycin D (7‐AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7‐AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7‐AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)‐fluorescence dual‐color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7‐AAD was used on fluoresceinisothiocyanate (FITC) and PE surface‐labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.

Analysis of flow cytometry histogram data by the subjective selection of an integration window can be a tedious and time‐consuming task and is often inaccurate. A new method for automated calculation of the percent positive from immunofluorescence histograms is presented. This new method is a modification of the currently used method of channel‐by‐channel histogram subtraction. Its accuracy is compared to that of the channel‐by‐channel histogram subtraction method and to another currently used automated method, which selects an integration window by finding the channels that contain the most fluorescent 2% of a control histogram.

The new histogram subtraction method is objective, easy to use, and is more accurate than other currently used automated analysis methods. PASCAL source code is given for each method of analysis.

A rapid, gentle, and sensitive method for quantification of cells undergoing apoptosis is presented. The method allows the simultaneous determination of dual‐color cell surface immunofluorescence. Cells are stained for 7 min with the vital dye Hoechst 33342 (HO342) for identification of live and apoptotic cells. 7‐amino‐actinomycin D (7‐AAD) is added to distinguish cells that have lost membrane integrity from apoptotic and live cells. Due to its spectral properties 7‐AAD can be utilized on cells that are dual‐surface labelled with fluoresceinisothiocyanate (FITC) and phycoerythrin (PE). The value of the method is demonstrated on human thymocytes, which constitutively undergo programmed cell death and which show an increase in the rate of apoptosis after exposure to the glucocorticoid dexamethasone (DEX). Vital staining with HO342 permits earlier detection of apoptotic changes compared to a staining technique in which cells are treated with a hypotonic citrate solution containing propidium iodide (PI) and the apoptotic cells are represented in a hypodiploid, “sub‐G₁” peak. The HO342/7AAD method may be particularly applicable to studies of programmed cell death in cells in which DNA fragmentation is difficult to detect by decreased DNA stainability. © 1994 Wiley‐Liss, Inc.

The energy transfer efficiency E was measured between fluorescein‐conjugated concanavalin A (Con A) and rhodamine‐conjugated Con A bound to homogeneous tissue culture cells, the HK22 murine lymphoma cell line. Results from a flow cytometric energy transfer method (FCET) and two different steady‐state fluorimeter methods were compared. The data were found to be in close agreement after careful correction of the steady‐state fluorimetric measurements for contributions from dissociating ligand. The biological variability of the individual cells with respect to E was calculated using an error propagation analysis and were found to be less than the variability in the absolute amount of ligand binding per cell. FCET has a number of advantages over the fluorimetric measurements using suspensions of cells: (1) relatively labile receptor‐ligand complexes can be measured; (2) the analysis can be restricted to undamaged cells by gating the data collection on the light‐scattering signals; (3) heterogeneous populations of cells with respect to donor and acceptor topology can be distinguished by the correlation of E with other cellular parameters derived from additional signals or combinations thereof; and (4) the dynamics of donor‐acceptor redistribution on subpopulations can be measured.

Flow cytometry has recently become a choice technique for the quantitative analysis of apoptosis. Monoparametric DNA analysis usually allows identification of apoptotic cells as a “subdiploid” peak. Progression through apoptosis leads to chromatin condensation, nuclear fragmentation and eventually to cell disruption. Thus, a major problem for the flow cytometric analysis of apoptotic populations is discrimination between debris and apoptotic cells. Here we demonstrate that the best parameter on which to make such a distinction is the DNA content, no matter what type of cell is studied. In contrast, discrimination between apoptotic, non‐apoptotic cells, and debris is possible on the basis of scattering signals only in few selected cases, depending on the morphology of the intact cells. © 1993 Wiley‐Liss, Inc.

Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub‐G₁ DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4/Thy‐1 dim thymocytes were included in the dye dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL‐60 cells treated with camptothecin. However, the dyes SYTO‐16 and LDS‐751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon‐ion laser. © 1995 Wiley‐Liss, Inc.

By halogenation of methylfluoresceindiacetate (MFDA) or eosin‐diacetate, two new dyes for cellular thiol compatible with visible laser excitation have become available. These probes circumvent the use of an ultraviolet (UV)‐excitation system as required by bimane‐based dyes and allow combination with probes for other cellular parameters. The thiol dyes attain maximal staining after 10 min at 37°C, and fluorescence is sensitive to pre‐treatment with diethylmaleate but not to buthionine sulfoximine. In a dual‐laser system, analysis of the cellular thiol level as a function of cell cycle distribution can be achieved in viable cells by simultaneous staining with the bisbenzimidazole dye Hoechst 33342 and one of the halogenated dyes. Using this approach, we were able to show that cells in the G2 phase of the cell cycle were more sensitive to thiol depletion with diethylmaleate than were cells in the G1 compartment. The new thiol dyes allow a more flexible selection of wavelengths of excitation and emission for assessing changes in cellular thiol (glutathione and other thiol compounds) and allow this parameter to be examined as a function of cell cycle position.