Here, we present a protocol for standardized high-content imaging and data processing of propidium iodide (PI)/Hoechst 33342-stained cells to perform reproducible cell viability analysis using automated image acquisition and analysis workflows.
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Method Article
Here, we present a protocol for standardized high-content imaging and data processing of propidium iodide (PI)/Hoechst 33342-stained cells to perform reproducible cell viability analysis using automated image acquisition and analysis workflows.
To address the challenges of low reproducibility due to operational variations in scientific research imaging and the complexity of analyzing a large number of samples using conventional methods, we introduce a protocol for staining HT22 cells with PI and Hoechst 33342 and then imaging and analyzing the data using the High-Content Imaging System. This workflow aims to establish robust imaging setting parameters and stringent quality control standards. It demonstrates the instrument's imaging settings and the workflow for three different image processing methods provided by the instrument: Multi-Wavelength Cell Scoring (Multi), Live-Dead (Live), and Custom Module Editor (CME). The image processing methods are compared and validated against the traditional method, ImageJ, to ensure their effectiveness. The study ultimately summarizes a workflow for using the High-Content Imaging System to determine cell viability using the PI and Hoechst double-staining method. By standardizing the entire imaging and data processing workflow, the study significantly enhances the reproducibility and reliability of scientific research imaging, thereby providing solid methodological support for cross-laboratory data comparison and advancing scientific research.
High-Content Imaging System serves as a crucial tool in modern scientific research, enabling high-speed imaging and multi-parameter assessment of injured or uninjured cells1,2,3. It plays an irreplaceable role in various disciplines. In the field of life sciences, it assists researchers in exploring the microscopic world of cells and tissues4,5. For instance, the precise detection of fluorescently labeled molecules and structures within cells allows clear visualization of cellular fine structures and dynamic changes
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This protocol uses a commercial cell line (HT22, ATCC) and does not involve human subjects or primary human tissues. No animals were used in this study.
1. Standardized sample preparation
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Impact of standardized preparation on imaging quality
The plate parameters were configured to ensure that the instrument could reliably locate the cell image layer. Samples prepared as a single layer of cells with moderate confluence facilitated image focusing at different objective magnifications and improved image recognition and quantitative analysis. Standardized sample preparation and optimized plate parameters effectively minimized edge effects, cell overlap, and other f.......
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Using the PI and Hoechst double staining method to determine cell viability, for example, the high-content instrument can quickly and in a high-throughput manner calculate cell viability within the same batch using built-in algorithm modules. This method has several advantages, including low sample volume, low cost, minimal cell damage, weak fluorescence bleaching, resistance to quenching, and the ability to capture multiple images. Additionally, the imaging time for a 96-well plate is generally only 30–45 min
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All authors declare that there is no conflict of interest. During the translation of the manuscript, Zhipu AI was used solely for translation and not for generating research data, experimental results, or core academic content. The authors assume full responsibility for the content of the manuscript.
The authors thank the support provided by the Chengdu University of Traditional Chinese Medicine School Foundation. The authors thank the support from the MPRC2022034 grant. This funding is used to develop a 3D dynamic imaging method for intestinal bacteria in Erchen Decoction to treat simple obese mice. The authors acknowledge the research platform provided by the Chengdu University of Traditional Chinese Medicine Innovative Institute of Chinese Medicine and Pharmacy and the Institute of Interdisciplinary Studies.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 1.5 mL Centrifuge Tube | Labigic Technology Co., Ltd. | MCT-001-150 | |
| 1000 µL Pipette Tips | Labigic Technology Co., Ltd. | 35423003E | |
| 15 mL Centrifuge Tube | Labigic Technology Co., Ltd. | CT-002-15A | |
| 2.5 µL Pipette Tips | Qingdao Haier Biomedical Co., Ltd. | B10T | |
| 200 µL Pipette Tips | Labigic Technology Co., Ltd. | T-001-200 | |
| 50 mL Centrifuge Tube | Labigic Technology Co., Ltd. | CT-002-50A | |
| 96 Well Cell Culture Plate | Corning Incorporated | 3599 | |
| Adjustable Single-Channel Pipette, 0.1–2.5 μL | Eppendorf SE | 3120000216 | |
| Adjustable Single-Channel Pipette, 100–1000 μL | Eppendorf SE | 3120000267 | |
| Adjustable Single-Channel Pipette, 20–200 μL | Eppendorf SE | 3120000259 | |
| Adjustable Single-Channel Pipette, 2–20 μL | Eppendorf SE | 3120000232 | |
| Cell Culture Dish | Corning Incorporated | 430167 | |
| Cryogenic Vials | Labigic Technology Co., Ltd. | BS-20-ST | |
| Dimethyl sulfoxide (DMSO) | Beijing Solarbio Science & Technology Co., Ltd. | D8371 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies Limited | 11966-025 | In this study, the complete DMEM was prepared using 10% FBS, 1% Penicillin-Streptomycin Solution, and 89% DMEM. |
| ESCO CelCulture CO2 Incubator | Esco Micro Pte. Ltd. | CCL-170B-8 | |
| Fetal Bovine Serum (FBS,Superfine) | Procell Life Science & Technology Co.,Ltd. | 164210-50 | |
| Hoechst 33342 Fluorescent Dyes | Beijing Solarbio Science & Technology Co., Ltd. | B8040 | |
| HT22 cell | Procell Life Science & Technology Co.,Ltd. | CL-0697 | |
| ImageJ | National Institutes of Health | ImageJ is one of the image processing software used in the manuscript. | |
| ImageXpress Micro Confocal High-Content Imaging System | Molecular Devices, LLC | ImageXpress Micro Confocal | In the manuscript, use "High-Content Imaging System" as a substitute for the instrument name. |
| Low speed freezing centrifuge | Hunan Xiangyi Laboratory Instrument Development Co., Ltd. | L530R | |
| MetaXpress High-Content Image Acquisition and Analysis Software | Molecular Devices, LLC | The software is a companion to the ImageXpress® Micro Confocal High-Content Imaging System. In the manuscript, "The analysis software provided with the device" refers to this software. | |
| OptiMair Vertical Laminar Flow Cabinet | Esco Micro Pte. Ltd. | ACB-4E1 | |
| Parafilm M | Pechiney Plastic Packaging Inc. | PM996 | |
| PBS | Shanghai Yuchun biology science and technology co., ltd | YC-5013 | |
| Penicillin-Streptomycin Solution | Procell Life Science & Technology Co.,Ltd. | PB180120 | |
| Propidium Iodide Solution (PI) | Beijing Solarbio Science & Technology Co., Ltd. | C0080 | |
| Trypsin-EDTA Solution | BasalMedia Technology Co.,Ltd. | S310KJ |
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