计算溶液所需的质量、体积或浓度。
YO-PRO-1/PI细胞凋亡与坏死检测试剂盒
有货
为什么选择此级别
BioReagent,无菌,适用于荧光分析 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
是否无菌：是
YO-PRO-1/PI 细胞凋亡与坏死检测试剂盒是一种基于 DNA 绿色荧光染料 YO-PRO-1（Oxazole yellow，YP1）和红色荧光染料碘化丙啶（Propidium Iodide，PI）的双荧光法检测细胞凋亡和坏死的试剂盒。
YO-PRO-1，又名恶唑黄（YP1），是一种非细胞膜穿透性的羰花青单体和 DNA 绿色荧光染料。在没有与 DNA 结合的时候基本上没有荧光，而与 DNA 结合后可以发出明亮的绿色荧光。在细胞凋亡发生时，细胞膜通透性发生改变，此时 YO-PRO-1 可以进入细胞内与 DNA 结合，发出明亮的绿色荧光。YO-PRO-1 结合 DNA 后的最大激发光波长为 491nm，最大发射波长为 509nm。
碘化丙啶（PI）是一种可对 DNA 染色的细胞核染色试剂。它是一种溴化乙啶的类似物，在嵌入双链 DNA 后释放红色荧光。PI 不能穿透完整细胞膜，但对凋亡晚期细胞和死细胞的破损细胞膜能够穿透，并使细胞核红染。PI-DNA 复合物的激发和发射波长分别为 535nm 和 617nm。
本试剂盒使用 YO-PRO-1 和 PI 配合用于分析和鉴定凋亡和坏死细胞，可以同时进行凋亡细胞和坏死细胞的检测，凋亡细胞呈现绿色荧光，坏死细胞同时呈现红色和绿色荧光阳性，活细胞很少或几乎没有荧光。本试剂盒作为一种细胞凋亡和坏死检测试剂盒，可以应用于大多数的哺乳动物细胞，包括贴壁细胞和悬浮细胞，与传统的 Annexin V-FITC/PI 检测方法相比，检测准确性和灵敏度相当，可适用于荧光显微镜、流式细胞仪、荧光酶标仪等荧光检测系统。
本试剂盒提供的 YO-PRO-1 和 PI 为 1000× 溶液，使用非常便捷。为了得到比较理想的染色结果，请根据细胞类型和实验实际情况对探针染色液的稀释倍数进行适当调整。YO-PRO-1 的工作浓度一般为 0.5-5×，推荐使用浓度为 1×；PI 的工作浓度为 0.1-4×，推荐使用浓度为 1×。
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使用方法
（一）染色工作液的配制（以 1000×稀释为例）：
由于不同细胞种类、细胞浓度的染色条件不同，建议自行摸索 YO-PRO-1 和 PI 的最适浓度。一般来说，满足信号足够的前提下，尽可能选择最低浓度的染料剂量。
根据样品数量和每个样品所需工作液的体积，计算出 YO-PRO-1/PI 染色工作液的总体积。对于 6、12、24、96 孔板，每孔 YO-PRO-1/PI 检测工作液的用量分别为 0.5~1 mL、200~500 μL、100~250 μL 和50~100 μL；对于流式细胞样品，每个样品的 YO-PRO-1/PI 染色工作液的体积为 0.5 mL。
下述按照 1:1000 稀释倍数 / 5mL 举例。
①取出 YO-PRO-1（1000×）、PI（1000×）和染色缓冲液，平衡至室温。
②在 5mL 的染色缓冲液中加入 5 µL 的 YO-PRO-1（1000×）和 5 µL 的 PI（1000×），涡旋震荡混匀制成工作液。
③所得到的工作液可直接用于染色细胞。
（二）荧光显微镜检测：
①接种培养。将细胞接种于 96 孔板等多孔板、细胞培养皿中或者细胞爬片上，按实验设计对细胞进行一定处理。
②洗涤。对于贴壁细胞，吸除培养液，用 PBS 洗涤细胞 1 次；对于悬浮细胞，250-1000×g 室温离心 5min，吸除上清，用 PBS 洗涤 1 次。酚红或血清对于本试剂盒的检测有一定的干扰。在能充分吸净残留液体的情况下，可以不使用 PBS 洗涤。
③染色。加入适当体积的 YO-PRO-1/PI 染色工作液。通常 96 孔板每孔加入 100 μL，24 孔板每孔加入 250 μL，12 孔板每孔加入 500 μL，6 孔板每孔加入 1 mL。37ºC 避光孵育 10-20min。不同的细胞最佳孵育时间有所不同，以 10 min 作为初始孵育时间，后续可以根据实际染色效果对染色时间进行适当调整和优化，以得到更加理想的染色效果。
④检测。孵育结束后，在荧光显微镜下观察荧光染色效果（YO-PRO-1 染色阳性细胞为绿色荧光，Ex/Em=491/509 nm；PI 染色阳性细胞为红色荧光，Ex/Em=535/617 nm）。如有需要，也可进一步进行其它荧光的复染，注意整个过程均需注意避光操作。
（三）流式细胞仪检测：
①细胞准备。贴壁细胞胰酶消化后用培养液重悬，并用 PBS 洗涤一次；悬浮细胞 250-1000×g 室温离心 5min，弃上清，用 PBS 洗涤一次。每个样品推荐的细胞用量为 10⁶个细胞。
②染色。对于上一步骤的 10⁶ 个细胞的沉淀，加入 0.5mL YO-PRO-1/PI 染色工作液，重悬为单细胞悬液。37℃避光孵育 20min。
注：需要准备好仅含缓冲液的细胞样品用作流式细胞仪检测时的阴性对照，该缓冲液与配制 YO- PRO-1/PI 检测工作液的缓冲液宜保持一致。同时准备两管额外的细胞样品，每管只加入一种染料（YO- PRO-1 或 PI），用于流式单染的补偿调节。
③检测。孵育完成后，可以直接进行流式细胞仪检测，也可以 250-1000×g 室温离心 5 min 沉淀细胞，吸净液体后每个样品加入 1mL 检测缓冲液重悬细胞后用流式细胞仪检测（YO-PRO-1 染色阳性细胞为绿色荧光，Ex/Em=491/509 nm；PI 染色阳性细胞为红色荧光，Ex/Em=535/617nm）。注意整个过程均需避光操作。染色后，尽量在 1 h 内进行流式细胞仪检测和分析。
注意：
1.使用仅含检测缓冲液的并且未经染色的细胞样品用于流式细胞仪的阴性对照设置。
2.细胞圈门时，注意不要圈入细胞碎片，并使用 YO-PRO-1 或 PI 单染的细胞进行调节补偿。双染细胞流式检测应获得两个相对独立的细胞群：①绿色荧光的凋亡细胞群；②红色荧光阳性坏死细胞群或红色荧光和绿色荧光双阳性的坏死细胞群。坏死细胞的绿色荧光染色通常会比较弱一些。
3.由于流式检测比较灵敏，使用的荧光探针浓度可能要比荧光显微镜检测时要低，此时可根据细胞类型和实际染色情况对 YO-PRO-1 或 PI 的稀释倍数进行适当调整。
（四）荧光酶标仪检测细胞死活的变化：
①接种培养。将细胞接种于 96 孔黑色多孔板中，每孔的细胞数需要控制在 100-10000 个，通常宜在2000-5000 个范围内。按实验设计对细胞进行一定处理。
②洗涤。对于贴壁细胞，吸除培养液，用 PBS 洗涤细胞 1 遍；对于悬浮细胞，250-1000×g 室温离心 5 min，吸除上清，用 PBS 洗涤 1 遍。酚红或血清对于本试剂盒的检测有一定的干扰。在能充分吸净残留液体的情况下，可以不使用 PBS 洗涤。
③染色。加入适当体积的 YO-PRO-1/PI 检测工作液，通常 96 孔板每孔加入 100 μL。37℃避光孵育5-20 min。不同的细胞最佳孵育时间有所不同，以 5 min 作为初始孵育时间，后续可以根据实际染色效果对染色时间进行适当调整和优化，以得到更加理想的染色效果。
④ 检测 。孵育结束后 ，用荧光酶标仪检测（ YO-PRO-1染色阳性细胞为绿色荧光 ，Ex/Em=491/509 nm；PI 染色阳性细胞为红色荧光，Ex/Em=535/617 nm）。通过对比对照组两种荧光探针的 RFU（Relative fluorescence values），可以得出凋亡细胞与坏死细胞数量的关系。
注意事项
1. 本试剂盒中的 YO-PRO-1 和 PI 量很少，有可能会粘在盖子或管壁上，开封前请先适当离心至管底。
2. 第一次使用前请将本品分装并于-20℃冻存，避免反复冻融。建议根据单次用量分装密封保存。
3. 染色工作液必须现配现用，配制好的工作液请在当天使用。荧光染料均存在淬灭问题，保存和使用本试剂盒的过程中请尽量注意避光，以减缓荧光淬灭。建议染色后尽量当天完成检测。
4. 染色缓冲液应在无菌环境中使用，否则可能会被微生物污染而影响使用效果，甚至无法继续使用。
5. 本试剂盒作用于某些细胞系流式检测时，YO-PRO-1 和 PI 双染后，可能会使 YO-PRO-1 出峰位置发生偏移，十字门可能需要根据实际情况重新划分。
6. YO-PRO-1 和 PI 对人体有刺激性，操作时请小心，并注意适当防护以避免直接接触人体或吸入体内。
7. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
8. 为了您的安全和健康，操作时请穿着实验服并佩戴一次性手套。
Is it sterile: Yes
This kit adopts a dual-fluorescence staining method based on the green fluorescent DNA dye YO-PRO-1 (Oxazole yellow, YP1) and the red fluorescent dye Propidium Iodide (PI) to detect cellular apoptosis and necrosis.YO-PRO-1, also known as Oxazole Yellow (YP1), is a membrane-impermeant monomeric carbocyanine nucleic acid stain with green fluorescence. It exhibits almost no intrinsic fluorescence in the free state, while it emits intense green fluorescence upon binding to DNA. During early apoptosis, the plasma membrane permeability changes, allowing YO-PRO-1 to penetrate into apoptotic cells and bind to intracellular DNA to produce bright green signals. The YO-PRO-1/DNA complex has a maximum excitation wavelength of 491 nm and a maximum emission wavelength of 509 nm. Propidium Iodide (PI) is a nuclear counterstain for DNA labeling and an ethidium bromide analog. It generates red fluorescence after intercalating into double-stranded DNA. PI cannot cross intact plasma membranes of viable cells, but it readily permeates the compromised membranes of late apoptotic and necrotic cells to stain cell nuclei red. The excitation and emission wavelengths of the PI-DNA complex are 535 nm and 617 nm, respectively. By combinatorial staining with YO-PRO-1 and PI, this kit enables simultaneous discrimination of apoptotic and necrotic cell populations. Apoptotic cells display positive green fluorescence only, necrotic cells are double-positive for both green and red fluorescence, and viable cells show negligible or no fluorescence signals. Compatible with most mammalian adherent and suspension cell lines, this kit delivers comparable accuracy and sensitivity to the classic Annexin V-FITC/PI assay platform. It is applicable to multiple fluorescence detection instruments, including flow cytometers, fluorescence microscopes and fluorescence microplate readers. Both YO-PRO-1 and PI supplied in the kit are formulated as 1000× stock solutions for convenient operation. To achieve optimal staining performance, the dilution factor of the fluorescent probes can be moderately optimized according to specific cell types and experimental conditions. The working concentration range of YO-PRO-1 is 0.5–5× with a recommended optimal concentration of 1×; the working concentration range of PI is 0.1–4× with a recommended optimal concentration of 1×.
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Operating Protocol
(I) Preparation of Staining Working Solution (Dilution at 1:1000)
Staining conditions vary greatly among cell types and cell concentrations. It is recommended to explore the optimal working concentrations of YO-PRO-1 and PI empirically. In general, use the lowest feasible dye concentration that yields acceptable fluorescent signals.
Calculate the total volume of YO-PRO-1/PI working solution according to the number of samples and required volume per well. Recommended volume per well for different plates:
6-well plate: 0.5–1 mL
12-well plate: 200–500 μL
24-well plate: 100–250 μL
96-well plate: 50–100 μL
For flow cytometry samples, prepare 0.5 mL working solution per single sample.
The following takes 5 mL total volume with 1:1000 dilution as an example:
1.Equilibrate YO-PRO-1 (1000× stock), PI (1000× stock) and staining buffer to room temperature.
2.Add 5 μL YO-PRO-1 (1000×) and 5 μL PI (1000×) into 5 mL staining buffer, vortex thoroughly to prepare the working solution.
3.The freshly prepared working solution is ready for cell staining.
(II) Detection via Fluorescence Microscope
1.Cell seeding and treatment
Seed cells in multi-well plates, cell culture dishes or cell slides, and apply experimental treatments as designed.
2.Washing step (optional)
For adherent cells: aspirate culture medium and rinse cells once with PBS.
For suspension cells: centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant and wash once with PBS.
Phenol red and serum will interfere with detection. Washing can be omitted if residual liquid can be fully removed.
3.Staining
Add appropriate volume of YO-PRO-1/PI working solution:
96-well plate: 100 μL/well
24-well plate: 250 μL/well
12-well plate: 500 μL/well
6-well plate: 1 mL/well
Incubate at 37 °C for 10–20 min in the dark. Optimal incubation time is cell-type dependent; start with 10 min and adjust according to actual staining effect for better results.
4.Imaging detection
Observe staining results under a fluorescence microscope immediately after incubation.
YO-PRO-1 positive apoptotic cells: green fluorescence, Ex/Em = 491 nm / 509 nm
PI positive late apoptotic/necrotic cells: red fluorescence, Ex/Em = 535 nm / 617 nm
Additional counterstaining is allowed if needed; keep samples protected from light throughout the whole procedure.
(III) Detection via Flow Cytometry
1.Cell preparation
Digest adherent cells with trypsin, resuspend in medium and wash once with PBS.
For suspension cells: centrifuge at 250–1000×g for 5 min at room temperature, remove supernatant and wash once with PBS.
Recommended cell number per sample: 1 × 10⁶ cells.
2.Staining
Resuspend the cell pellet in 0.5 mL YO-PRO-1/PI working solution to form a single-cell suspension. Incubate at 37 °C for 20 min protected from light.
Notes:
Prepare an unstained cell control suspended in identical staining buffer as the negative control.
Prepare two extra single-stain tubes (only YO-PRO-1 or only PI) for fluorescence compensation adjustment.
3.Instrument analysis
Samples can be tested directly after incubation. Alternatively, pellet cells by centrifugation at 250–1000×g for 5 min at room temperature, remove supernatant, resuspend in 1 mL detection buffer for flow cytometry analysis.
YO-PRO-1: Ex/Em = 491/509 nm (green channel)
PI: Ex/Em = 535/617 nm (red channel)
Complete flow acquisition and analysis within 1 hour post staining, and avoid light exposure at all steps.
Important Notes for Flow Cytometry
1.Use unstained cells suspended in detection buffer to set up negative control gates.
2.Exclude cell debris during gating. Apply single-stained samples for compensation calibration. Two distinct populations should be resolved in dot plots:
Early apoptotic cells: only YO-PRO-1 green positive
Necrotic/late apoptotic cells: PI single positive or double positive (weak green signal for YO-PRO-1 is commonly seen in necrotic cells)
3.Due to high sensitivity of flow cytometry, lower probe dilution ratios may be required compared with microscope detection; optimize the dilution factor based on cell lines and staining performance.
(IV) Detection of Cell Viability Change via Fluorescence Microplate Reader
1.Cell seeding
Seed cells in black 96-well plates, with cell density ranging from 100 to 10,000 cells per well (optimally 2000–5000 cells/well). Conduct subsequent experimental treatments.
2.Washing step (optional)
Adherent cells: discard medium and wash once with PBS.
Suspension cells: centrifuge at 250–1000×g for 5 min at room temperature, remove supernatant and wash once with PBS.
PBS washing can be skipped if residual liquid is thoroughly aspirated, as phenol red and serum cause background interference.
3.Staining
Add 100 μL YO-PRO-1/PI working solution per well for 96-well plates. Incubate at 37 °C in the dark for 5–20 min. Initiate incubation at 5 min and fine-tune the duration according to practical staining outcomes.
4.Plate reading
Measure fluorescence intensity with a microplate reader:
YO-PRO-1: Ex/Em=491 nm/509 nm
PI: Ex/Em=535 nm/617 nm
Quantify the relative population ratio of apoptotic and necrotic cells by comparing RFU (Relative Fluorescence Units) values between treatment groups and blank controls.
Precautions
1.The volume of YO-PRO-1 and PI stock solutions is limited; brief centrifugation before opening the vial to spin down liquid attached on cap or tube wall.
2.Aliquot the stock dyes upon first receipt and store at −20 °C to avoid repeated freeze-thaw cycles. Seal aliquots according to single experimental dosage for long-term preservation.
3.Prepare working solution freshly right before use and finish staining on the same day. Fluorophores are prone to photobleaching; store and operate the kit under dark conditions, and complete detection immediately after staining.
4.Staining buffer must be used under sterile conditions to prevent microbial contamination, which will compromise assay performance or render the buffer unusable.
5.For certain cell lines analyzed by flow cytometry, YO-PRO-1 fluorescence peak may shift after double staining with PI; redefine the quadrant gates according to actual dot plot distribution.
6.YO-PRO-1 and PI are irritant to human body. Wear appropriate PPE to prevent direct skin contact and inhalation during manipulation.
7.This product is for research use only. Not for clinical diagnosis, therapeutic administration, food or pharmaceutical production. Do not store in residential premises.
8.Always wear a lab coat and disposable gloves for personal safety during all experimental operations.
规格
别名
流式细胞凋亡坏死双染试剂盒（YO-PRO-1/PI）
英文别名
YO-PRO-1/PI Apoptosis and Necrosis Detection Kit
规格或纯度
BioReagent, 适用于荧光分析, 无菌
英文名称
YP1/PI Apoptosis and Necrosis Detection Kit
应用
Apoptosis Assay
样品类型
细胞
检测方法
荧光法
定量&半定量
半定量
检测仪器
流式细胞仪,荧光显微镜,荧光酶标仪
检测波长
YO‑PRO‑1 (Ex/Em: 491/509 nm) PI (Ex/Em: 535/617 nm)
敏感性
Light-sensitive
Emission(nm)
509nm, 617nm
Excitation(nm)
491nm, 535nm
Ex/Em(nm)
Ex:535nm;Em:617nm,Ex:491nm;Em:509nm
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
稳定性与储存
Store at -20℃ long term (12 months). Store in the dark.
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