Screening for Apoptosis and Cellular Necrosis Using the SpectraMax Paradigm Multi-Function Detection Platform - Molecular Devices

2022-11-12 04:04:56

Introduction

The nature of both apoptosis and necrosis is cell death, but we can distinguish it by its characteristics and pathological morphology. Apoptosis refers to programmed cell death and no cell membrane damage; cell necrosis is thought to be passive death due to pathology, accompanied by cell membrane damage, resulting in the release of cytoplasmic factors and inflammatory responses. In the screening process of anti-tumor drugs, it is very important to distinguish between cell apoptosis and cell necrosis, because the main side effect of the latter is to cause inflammatory reactions of cells. Here we describe the use of SpectraMax Paradigm (Molecular Devices) and Vybrant Apoptosis Detection Kit (Invitrogen) for this study.

Test description

The Vybrant Apoptosis Detection Kit is based on the two markers of Annexin V and Sytox Green , which can quickly and easily distinguish between apoptosis and necrosis of tumor cells. Annexin V is a human vascular anticoagulant that is commonly used to detect the occurrence of apoptotic cells after labeling phycoerythrin (R-PE). The working principle is that in normal cells, phosphatidylserine is only distributed in the inner side of the lipid bilayer of the cell membrane. When the cell undergoes apoptosis, the membrane phosphatidylserine (PS) is turned from the inside to the outside of the lipid membrane. Annexin V is a kind of Phospholipid binding protein with high affinity for phosphatidylserine. In contrast, Sytox Green dyes do not penetrate into living cells and apoptotic cells, but can enter necrotic cells and stain their nuclei, emitting strong green fluorescence. We planted tumor cells in 384-well plates in advance, and then used tumor necrosis factor (TNF) and actinomycin D (Actinomycin-D) to induce apoptosis, using polyethylene glycol octyl phenyl ether ( Triton). X-100 ) induces cell necrosis. In addition, we can distinguish between apoptosis and necrosis by detecting the anti-tumor properties and differentiation characteristics of different substances expressed by cell tissues.

Use different detection methods to detect apoptosis and necrosis of tumor cells

SpectraMax Paradigm is a new concept multi-function detection platform launched by Molecular Devices of the United States (Figure 1). It separates the light source system and the detection system independently. Multiple sets of detectors are fixed on the host at the same time. Different card boxes are selected for different experimental requirements. Each type of card box is actually an independent optical path system, which can improve the detection and sensitivity of detection. . According to the characteristics of this test, if the instrument can detect the fluorescence intensity of both Annexin V and Sytox Green markers, the detection efficiency can be greatly improved (Fig. 2), and Paradigm's dual-channel detector can work at the same time, which is very suitable. Such test requirements.

Figure 1: SpectraMax Paradigm Multi-Function Detection Platform

Figure 2. HepG2 cells were seeded in flat-bottomed 384-well plates 24 hours in advance. Some of these wells were left untreated, while another part of the cells was treated with TNF (25 ng/ml) and Actinomycin-D 18 hours before the test. (1 ug/ml) was incubated with Triton X-100 (0.1%), then the medium in the microplate was removed, and the two marker molecules were dissolved in buffer and incubated at 37 degrees for 20 minutes. After staining, the results of the single-point (a), on-the-fly (b) and area-scan (c) modes of the SpectraMax Paradigm multi-detection platform were used , and each column height represents the average fluorescence intensity value (RFU). ) and the standard deviation of 5 replicate wells. It can be seen that the larger the area inside the scanning hole, the more accurate the result is, especially suitable for the detection test of single-layer heterogeneous cells like tumor cells.

in conclusion

In summary, we can find that SpectraMax Paradigm is an ideal detection platform based on cytology application. It is found from the results of Vybrant apoptosis detection kit that the cell layer in the well changes very quickly and affects the whole test result. Big, Paradigm's "On-the-fly" data collection mode can solve this problem well. The high-speed dual-emission detector draws a linear plot of the multiple data points measured for each well, resulting in a larger measurement area value that will result in better statistical results. The use of the "area scan" function further enhances the quality of experimental data based on cytology applications. In addition, the dual-channel simultaneous detection function of this instrument can greatly shorten the detection time and improve the efficiency of screening work.

Acknowledgement

I am very grateful to the research data provided by the Medical Biotechnology Department of the Krems High School of Higher Education in Austria.

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