SYTOX Green死细胞染料 (用于流式细胞分析) 是专用于流式细胞术、特异性标记膜破损坏死细胞细胞核的绿色荧光探针试剂。其核心作用原理为：SYTOX Green 无法穿透结构完整的健康活细胞膜，仅能自由进入细胞膜受损的晚期凋亡、坏死细胞内部，与胞内双链 DNA 高亲和力结合，游离状态下染料几乎无荧光；该染料发射光谱干扰极小，相邻荧光通道补偿需求低；染料与核酸结合后荧光强度提升 500 倍以上，是操作简便、可定量的单步死细胞指示剂。在 488 nm 流式通用氩离子激光激发下，于 525 nm 波段产生明亮绿色荧光，实现活、死细胞清晰分群。
对比 PI、7-AAD 等传统死细胞染料，本品具备多重突出优势：
(1) 荧光亮度更高、死活细胞信号差距显著，低比例死细胞也可精准检出；
(2) 单步染色免洗涤流程，减少珍贵原代、微量细胞离心损耗；
(3) 本品核心应用场景覆盖流式细胞术细胞活力定量；可搭配 Annexin V 系列荧光偶联物同步染色，一次性区分活细胞、早期凋亡细胞、坏死细胞；可与活细胞核酸染料联合使用，实现活死细胞双色同步区分；广泛用于药物细胞毒性筛选、凋亡机制评估、免疫细胞杀伤实验、干细胞复苏质控、微生物药敏检测等细胞生物学基础研究。
精准判定细胞活力、区分凋亡与坏死是细胞免疫学、肿瘤药理学、微生物学研究的基础前提，细胞膜完整性是界定细胞存活状态的核心金标准，传统台盼蓝计数染色存在灵敏度不足、多色实验受限，无法满足高通量、高参数流式精准定量需求，SYTOX Green 死细胞染料基于膜通透性差异荧光标记技术应运而生，依托 488 nm 激发、525 nm 发射的经典绿光通道，完美匹配市面全部主流流式仪器，填补了多色流式体系高效死细胞分群的技术空白。在各类科研场景中，在抗肿瘤药物、纳米材料筛选中，可稳定量化细胞死亡比例，获得剂量 - 毒性相关性定量数据；在干细胞、原代免疫细胞分离与体外培养质控中，快速评估细胞复苏存活率，筛选高活性细胞群体；在多色免疫分型、单细胞测序前样本质控实验中，兼容多荧光抗体面板，同步完成细胞亚群分群与活力质控。该试剂为细胞存活、凋亡、毒性相关研究提供稳定、可重复的定量检测手段，大幅简化流式实验操作流程、缩短样本处理时长，高效产出精准可靠的流式数据，助力细胞死亡调控机制、肿瘤靶向药物研发、免疫治疗机理、微生物耐药机制等基础科研开展，同时为体外细胞药效评价体系搭建标准化检测方案，推动基础细胞研究成果向体外临床转化研究落地。
应用范围：凋亡联合检测、细胞毒性检测、细胞活力监测、氧化应激、冻存、药物刺激等细胞损伤机制研究。
产品特点：
● 操作简便高效：一步染色免洗涤，减少珍贵细胞损耗，室温避光孵育即可上机；
● 区分效果优异：仅穿透膜破损死细胞，结合核酸后荧光强度提升 500 倍以上，死活细胞分群清晰，低死亡率样本也可精准检出；
● 多色兼容性强：光谱干扰小，可搭配 Annexin V、以及各类远红 / 紫光荧光素搭建多色流式方案；
● 信号稳定重复性好：染色均匀、背景低，荧光信号稳定，实验结果可靠；
● 应用场景丰富：支持细胞活力、凋亡、细胞周期、药物毒性、免疫杀伤、单细胞测序前质控等多种实验。
产品参数：
● Ex/Em：488/525 nm
注意事项：
● 使用次数为 1 mL 细胞悬液加入 1 μL SYTOX Green。
● 使用前请将产品瞬时离心，再进行后续操作。收到时可以根据需要适当进行分装。
● 荧光染料均存在淬灭问题，请尽量注意避光。
● 为了您的安全和健康，请遵循您所在常规实验室安全规定。
● 本产品仅限于科研用途并且不得存放于普通住宅内。
使用说明：
一、实验前准备
1. 试剂准备：
● 将试剂从储存条件（如 4 ℃）取出，平衡至室温 (15~25 ℃)，准备足量的 HBSS/PBS。
注：优先选用无磷酸盐缓冲体系进行染色。
2. 仪器准备：
● 流式细胞仪：激发 / 发射波长 Ex/Em：488/525 nm。
二、操作步骤
方案一：流式检测
1. 收集细胞悬液至离心管中。
2. 使用 HBSS 重悬细胞沉淀。调整细胞浓度至 1×10⁵~5×10⁷个 /mL。
3. 每支流式管加入 1 mL 细胞悬液。每管加入 1 μL SYTOX Green，充分吹打混匀。
4. 室温避光孵育最低 15 min，孵育时长可根据实际观察到的染色效果进行灵活调整，调整区间为 5~30 min。
注：各染料的孵育时长、加样量可以根据实验所用的不同细胞种类、预实验结果或实验条件设置的使用量进行调整。
5. 无需洗涤，可直接上机检测。采用 488 nm 激光激发，530/30 滤光片（或等效滤片）采集荧光信号。
注：染色后建议 1 小时内完成上机检测。
方案二：联合检测方案
注：本产品可以与 Annexin V 荧光偶联物搭配使用，一次性区分活细胞、凋亡细胞、坏死细胞。
1. 先按照其他荧光染料说明书进行染色、洗涤步骤。
2. 待其他荧光染料染色完成后。按照方案一进行 SYTOX Green 的染色。
常见疑问与解答：
● 问：为什么完整活细胞不会被染色？
答：该染料为膜不通透型染料，无法穿透结构完整的活细胞膜；仅细胞膜破损时才能进入细胞结合 DNA 产生荧光。
● 问：这个可以用来对冰冻切片和细胞爬片染色吗？
答：细胞爬片是可以的，冰冻切片目前没有测试经验。
SYTOX Green Dead Cell Stain for Flow Cytometry is a green fluorescent probe reagent specifically designed to label the nuclei of dead cells with compromised membranes. Its core working principle is as follows: SYTOX Green cannot penetrate intact, healthy live cell membranes; it only freely enters late apoptotic and necrotic cells with damaged cell membranes and binds with high affinity to intracellular double‑stranded DNA. The dye exhibits almost no fluorescence in its free state. It produces minimal spectral interference and requires low compensation for adjacent fluorescence channels. Fluorescence intensity increases more than 500‑fold upon binding to nucleic acids, making it a convenient, quantifiable one‑step indicator for dead cells. Excited by the common 488 nm argon ion laser for flow cytometry, it emits bright green fluorescence at 525 nm to achieve clear separation of live and dead cell populations.
Compared with traditional dead cell dyes such as PI and 7‑AAD, this product has multiple prominent advantages:
(1) Higher fluorescence brightness and a distinct signal gap between live and dead cells, enabling accurate detection even in samples with a low proportion of dead cells;
(2) One‑step staining with no washing required, reducing centrifugation‑induced loss of precious primary cells and trace cell samples;
(3) Its core applications include flow cytometric quantification of cell viability. It can be co‑stained with Annexin V fluorophore conjugates to simultaneously distinguish live cells, early apoptotic cells and necrotic cells. It can also be combined with live‑cell nucleic acid dyes for dual‑colour live/dead cell discrimination. It is widely used in drug cytotoxicity screening, apoptosis mechanism evaluation, immune cell killing assays, quality control for stem cell recovery, microbial susceptibility testing and other fundamental cell biology studies.
Accurate determination of cell viability and discrimination between apoptosis and necrosis are fundamental prerequisites for research in cellular immunology, tumour pharmacology and microbiology. Cell membrane integrity serves as the gold standard for defining cell viability. Traditional trypan blue staining suffers from insufficient sensitivity and limited compatibility with multicolour assays, failing to meet the demand for high‑precision quantification in high‑throughput, high‑parameter flow cytometry. SYTOX Green dead cell dye was developed based on differential membrane permeability fluorescent labelling technology. Utilising the classic green channel with 488 nm excitation and 525 nm emission, it is fully compatible with all mainstream flow cytometers on the market and fills the technical gap for efficient dead‑cell population separation in multicolour flow cytometry systems. In various research scenarios, it can stably quantify the proportion of cell death in anti‑tumour drug and nanomaterial screening to obtain quantitative dose‑toxicity correlation data. For quality control during isolation and in‑vitro culture of stem cells and primary immune cells, it rapidly assesses post‑thaw cell viability and selects high‑viability cell populations. In multicolour immunophenotyping and pre‑sequencing sample QC for single‑cell sequencing, it is compatible with multiplex fluorescent antibody panels to complete cell subset gating and viability assessment simultaneously. This reagent provides a stable, reproducible quantitative detection method for research related to cell survival, apoptosis and cytotoxicity. It greatly simplifies the workflow of flow cytometry experiments, shortens sample processing time and generates accurate and reliable flow data efficiently. It supports fundamental research on cell death regulatory mechanisms, targeted anti‑tumour drug development, immunotherapy mechanisms and microbial resistance mechanisms. Meanwhile, it establishes a standardized detection protocol for in‑vitro cellular pharmacodynamic evaluation systems and promotes the translational application of fundamental cellular research findings toward in‑vitro clinical studies.
Applications
Combined apoptosis detection, cytotoxicity assays, cell viability monitoring, oxidative stress, cryopreservation, drug stimulation and other studies on cellular damage mechanisms
Product Features
● Simple and efficient operation: one‑step staining without washing, minimizing loss of precious cells; ready for instrument acquisition after incubation at room temperature protected from light.
● Excellent discrimination performance: penetrates only dead cells with damaged membranes; fluorescence intensity increases more than 500‑fold after nucleic acid binding, live and dead cell populations are well separated, and samples with low mortality can be accurately detected.
● High multiplex compatibility: low spectral crosstalk; compatible with Annexin V and various far‑red / violet fluorophores for designing multicolour flow cytometry panels.
● Stable signal with good reproducibility: uniform staining, low background, stable fluorescent signals and reliable experimental results.
● Rich application scenarios: compatible with cell viability, apoptosis, cell cycle, drug toxicity, immune killing, pre‑QC for single‑cell sequencing and other assays.
Product Specifications
● Ex/Em: 488/525 nm
Precautions
● Recommended usage: add 1 μL of SYTOX Green to 1 mL of cell suspension.
● Briefly centrifuge the vial before use. Aliquot the product as needed upon receipt.
● Fluorescent dyes are susceptible to photobleaching. Keep the product protected from light as much as possible.
● Follow standard laboratory safety regulations for your personal safety and health.
● For research use only. Do not store this product in residential premises.
Instructions for Use
Ⅰ Pre‑experiment Preparation
1. Reagent Preparation
● Remove the reagent from storage (e.g. 4 °C) and equilibrate it to room temperature (15–25 °C). Prepare sufficient HBSS / PBS.
Note: Phosphate‑free buffer systems are preferred for staining.
2. Instrument Preparation
● Flow cytometer: Excitation / Emission wavelength Ex/Em: 488/525 nm.
Ⅱ Operating Procedures
Protocol 1: Flow Cytometry Detection
1. Transfer the cell suspension into centrifuge tubes.
2. Resuspend the cell pellet with HBSS. Adjust the cell concentration to 1×10⁵ ~ 5×10⁷ cells/mL.
3. Add 1 mL of cell suspension to each flow tube, followed by 1 μL of SYTOX Green, and mix thoroughly by pipetting.
4. Incubate at room temperature protected from light for a minimum of 15 min. The incubation time can be adjusted flexibly from 5 to 30 min according to the actual staining results.
Note: Incubation duration and dosage can be adjusted based on cell type, preliminary test results or experimental settings.
5. No washing is required; samples can be analysed directly on the instrument. Use 488 nm laser excitation and collect fluorescent signals using a 530/30 bandpass filter (or equivalent filter).
Note: It is recommended to complete instrument acquisition within 1 hour after staining.
Protocol 2: Co‑staining Assay
Note: This product can be used together with Annexin V fluorophore conjugates to distinguish live cells, apoptotic cells and necrotic cells in a single run.
1. Perform staining and washing steps following the instructions for other fluorescent dyes first.
2. After staining with other fluorescent dyes is completed, stain with SYTOX Green following Protocol 1.
FAQ
Q: Why are intact live cells not stained?
A: This is a membrane‑impermeant dye. It cannot cross intact live cell membranes. It can only enter cells and bind DNA to produce fluorescence when the cell membrane is compromised.
Q: Can this dye be used for frozen sections and cell coverslip staining?
A: It is suitable for cell coverslips. It has not been validated for frozen sections.
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