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Sep 2015

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Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers
DNA和有丝分类marker同步染色后利用流式细胞术快速分析细胞周期   

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Abstract

The flow cytometric quantitation of DNA content by DNA-binding fluorochrome, propidium iodide (PI) is the most widely used method for cell cycle analysis. However, the commonly used methods are time-consuming and labor-intensive and are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Here, we report an optimized simple protocol for rapid and simultaneous analysis of characteristic mitotic phosphorylated proteins and DNA content, permitting the quantification of cells in mitosis, G1, S and G2 stage in a single assay. The protocol detailed here employs detergent-based hypotonic solution to rapidly permeabilize cells and allows simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3, with specific antibody within 20 min. The protocol requires only inexpensive and commercial available reagents and also enables a rapid and complete analysis of cell cycle profile.

Keywords: Cell cycle (细胞周期), Mitosis (有丝分裂), Hypotonic buffer (低渗缓冲液), Propidium iodide (碘化丙啶), Histone H3 (组蛋白H3)

Background

Cell cycle analysis by flow cytometry is mainly based on measurement of DNA content by stain with propidium iodide (PI). The stoichiometric nature of PI ensures the accurate quantification of DNA content and allows us to reveal the distribution of cells in G1, S and G2 cell cycle stage or in sub-G1 cell death stage, the latter of which is characterized by DNA fragmentation. Most of the commonly used methods for PI-based DNA quantification require fixation using alcohol or aldehyde, which is time-consuming and labor-intensive. In addition, these methods are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Therefore, we adopted and optimized a previous established hypotonic buffer to permeabilize cells, allowing simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3 (pH3), with pH3 specific antibody (Riccardi et al., 2006; Liu et al., 2016). This method enables a rapid (within 20 min) and comprehensive analysis of cell cycle profile (G1, S, G2 and M phase).

Materials and Reagents

  1. Falcon® 5 ml round bottom polystyrene test tube (Corning, Falcon®, catalog number: 352058 )
  2. Dulbecco’s phosphate buffered saline, no calcium, no magnesium (DPBS) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 14200166 )
  3. Alexa Fluor® 647 rat anti-phospho-Histone H3 (pS28) (BD, BD Bioscience, catalog number: 558217 )
  4. Sodium citrate (Na3C6H5O7·2H2O) (Sigma-Aldrich, catalog number: 1613859 )
  5. TritonTM X-100 (Sigma-Aldrich, catalog number: T8787 )
  6. Propidium iodide (PI) (C27H34I2N4) (Sigma-Aldrich, catalog number: P4170 )
  7. Hypotonic lysis/PI buffer (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5424 R )
  2. Flow cytometer (ACEA BIO, model: NovoCyte Flow Cytometer , 488-nm laser line, for excitation)

Software

  1. FlowJo_V10 is used for analyzing the flow cytometry data

Procedure

Note: All the procedures are done at room temperature.

  1. Suspend cells at 0.4 x 106 cells in 1 ml of DPBS in Falcon® 5 ml tubes.
  2. Centrifuge at 200 x g for 5 min.
  3. Aspirate off the DPBS.
  4. Re-suspend the cell pellet in 100 μl of hypotonic buffer, add 0.5 μl Alexa Fluor® 647 anti-phospho-Histone H3 per sample (Alexa Fluor 647 can be detected at APC channel), then gently mix the sample.
    Note: Since the hypotonic shock removes the majority of the RNA, RNase treatment is not required.
  5. Place the tubes in the dark at room temperature, before flow cytometry analysis, for at least 20 min but no longer than 2 h.
    Note: Including an additional washing step before measurement does not change the result but may cause cell loss.
  6. Measure fluorescence (detector 585/40 nm at PE channel for PI and 675/30 nm at APC channel for phospho-Histone H3 staining) by flow cytometer. Collect at least 20,000 total events, gate-out residual debris and measure diploid and tetraploid DNA peaks (Figures 1A-1D).


    Figure 1. Illustration of the gating strategy in analyzing cell cycle profile of HeLa cell. A. Exclusion of cell debris by forward scatter (FSC) and side scatter (SSC); B. Exclusion of clumps and doublets by forward scatter width (FSC-W) and height (FSC-H); C. Identification of G1, S and G2/M phase by PI; D. Identification of M-phase by mitosis-specific antibody (phospho-Histone H3-APC). The final percentage (%) of cells for each phase is about G1: 61.6%, S: 13.8%, G2/M: 22.5, M: 1.59% according to the analysis.

Data analysis

As shown in Figure 1, the histogram of DNA content could categorize cells into three groups, G1, S and G2/M phase (Figure 1C). Mitosis-specific antibody (phospho-Histone H3-APC) is applied to distinguish G2 from M cells (Figure 1D).

Notes

  1. For applying this protocol to other cell lines, the careful titration on cell number is recommended.
  2. Leaving the cell in hypotonic buffer for longer than 2 h may significantly increase the cell debris.
  3. Hypotonic buffer is not compatible with concurrent examination of intracellular fluorescent (GFP, RFP etc.) fusion proteins.

Recipes

  1. Hypotonic lysis/PI buffer
    Deionized/distilled water
    0.1% sodium citrate (wt/v)
    0.1% Triton X-100 (v/v)
    50 μg/ml PI
    Notes:
    1. Solution can be kept at 4 °C for months.
    2. PI is a suspected carcinogen and should be handled with care. The dye must be disposed of safely and in accordance with applicable local regulations.

Acknowledgments

This work was supported by R21AI117547 and 1R01AI114581 from National Institute of Health, V2014-001 from the V-Foundation and 128436-RSG-15-180-01-LIB from the American Cancer Society (R.W.).

References

  1. Liu, L., Lu, Y., Martinez, J., Bi, Y., Lian, G., Wang, T., Milasta, S., Wang, J., Yang, M., Liu, G., Green, D. R. and Wang, R. (2016). Proinflammatory signal suppresses proliferation and shifts macrophage metabolism from Myc-dependent to HIF1α-dependent. Proc Natl Acad Sci U S A 113(6): 1564-1569.
  2. Riccardi, C. and Nicoletti, I. (2006). Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1(3): 1458-1461.

简介

DNA结合荧光染料,碘化丙啶(PI)对DNA含量的流式细胞定量是细胞周期分析中使用最广泛的方法。 然而,常用的方法是耗时且劳动密集的,并且与通过荧光标记的抗体对有丝分裂标记物的染色不相容。 在这里,我们报告了一个优化的简单方案,用于快速和同时分析特征有丝分裂磷酸化蛋白和DNA含量,允许在单次测定中有丝分裂,G1,S和G2期细胞的定量。 这里详述的方案采用基于洗涤剂的低渗溶液来快速透化细胞,并允许在20分钟内同时用PI和有丝分裂标记物磷酸组蛋白H3与特异性抗体染色DNA。 该方案仅需要廉价和商业可用的试剂,并且还能够快速和完整地分析细胞周期特征。
【背景】通过流式细胞术进行细胞周期分析主要基于用碘化丙啶(PI)染色测量DNA含量。 PI的化学计量特性确保DNA含量的准确定量,并且允许我们揭示G1,S和G2细胞周期阶段或G1期细胞死亡期细胞的分布,后者以DNA片段化为特征。用于基于PI的DNA定量的大多数常用方法需要使用酒精或醛固色,这是耗时且劳动密集型的。此外,这些方法与通过荧光标记的抗体对有丝分裂标记物的染色不相容。因此,我们采用和优化了以前建立的低渗缓冲液以使细胞透化,允许用PI和有丝分裂标记物磷酸组蛋白H3(pH3)同时染色DNA与pH3特异性抗体(Riccardi等人,2006; Liu et al。 ,2016)。该方法能够快速(20分钟内)并对细胞周期分布(G1,S,G2和M期)进行综合分析。

关键字:细胞周期, 有丝分裂, 低渗缓冲液, 碘化丙啶, 组蛋白H3

材料和试剂

  1. Falcon ® 5 ml圆底聚苯乙烯试管(Corning,Falcon ®,目录号:352058)
  2. Dulbecco的磷酸盐缓冲盐水,不含钙,无镁(DPBS)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:14200166)
  3. Alexa Fluor大鼠抗磷酸组蛋白H3(pS28)(BD,BD Bioscience,目录号:558217)
  4. 柠檬酸钠(Na 3 C 6 H 5 O 7 / 2H 2 O) )(Sigma-Aldrich,目录号:1613859)
  5. Triton TM X-100(Sigma-Aldrich,目录号:T8787)
  6. 碘化丙啶(PI)(C 27 H 34 N 12 N 4)(Sigma-Aldrich,目录号:P4170)
  7. 低渗裂解/ PI缓冲液(参见食谱)

设备

  1. 离心机(Eppendorf,型号:5424 R)
  2. 流式细胞仪(ACEA BIO,型号:NovoCyte Flow Cytometer,488nm激光线,用于激发)

软件

  1. FlowJo_V10用于分析流式细胞仪数据

程序

注意:所有程序都在室温下完成。

  1. 将悬浮细胞在0.4×10 6个细胞中的1ml DPBS中,在Falcon的5ml管中。
  2. 以200 x g离心5分钟。
  3. 吸引DPBS。
  4. 将细胞沉淀重新悬浮在100μl低渗缓冲液中,每个样品加入0.5μlAlexa Fluor 6470抗磷酸组蛋白H3(Alexa Fluor 647可在APC通道检测),然后轻轻混合样本。
    注意:由于低渗休克消除了大部分RNA,因此不需要RNase治疗。
  5. 将管置于室温下,在流式细胞仪分析前至少20分钟,但不超过2小时。
    注意:在测量之前包括额外的洗涤步骤不会改变结果,但可能导致细胞丢失。
  6. 通过流式细胞仪测量荧光(PI通道检测器585/40 nm,APC通道675/30 nm磷酸组蛋白H3染色)。收集至少20,000次事件,门禁残留碎片并测量二倍体和四倍体DNA峰(图1A-1D)。


    图1.分析策略在分析HeLa细胞的细胞周期谱中的图示。A.通过前向散射(FSC)和侧向散射(SSC)排除细胞碎片; B.通过前向散射宽度(FSC-W)和高度(FSC-H)排除团块和双峰; C.通过PI识别G 1,S和G 2 / M相; D.通过有丝分裂特异性抗体(磷酸组蛋白H3-APC)鉴定M期。每个相的细胞的最终百分比(%)约为G <1:61.6%,S:13.8%,G 2 / M:22.5,M:1.59%分析。

数据分析

如图1所示,DNA内容的直方图可以将细胞分为三组,G 1,S和G 2 / M / M期(图1C)。应用有丝分裂特异性抗体(磷酸组蛋白H3-APC)来区分M细胞的G 2亚(图1D)。

笔记

  1. 为了将此协议应用于其他细胞系,建议小心滴定细胞数
  2. 将细胞置于低渗缓冲液中超过2小时可能会显着增加细胞碎片。
  3. 低渗缓冲液与细胞内荧光(GFP,RFP等)融合蛋白的同时检测不兼容。

食谱

  1. 低音裂解/ PI缓冲液
    去离子/蒸馏水
    0.1%柠檬酸钠(wt / v)
    0.1%Triton X-100(v / v)
    50μg/ ml PI
    注意:
    1. 解决方案可以在4°C下保存数月。
    2. PI是一种疑似致癌物质,应小心处理。染色剂必须安全处理,并符合适用的当地法规。

致谢

这项工作得到了国立卫生研究院的R21AI117547和1R01AI114581,V-Foundation的V2014-001和美国癌症协会(R.W.)的128436-RSG-15-180-01-LIB的支持。

参考

  1. Liu,L.,Lu,Y.,Martinez,J.,Bi,Y.,Lian,G.,Wang,T.,Milasta,S.,Wang,J.,Yang,M.,Liu, Green,DR和Wang,R。(2016)。促炎信号抑制增殖并将巨噬细胞代谢从Myc依赖性转移到HIF1α依赖性。 Proc Natl Acad Sci USA 113(6):1564-1569。
  2. Riccardi,C.和Nicoletti,I.(2006)。通过碘化丙啶染色和流式细胞术分析细胞凋亡 Nat Protoc 1(3):1458-1461。
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引用:Shen, Y., Vignali, P. and Wang, R. (2017). Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers. Bio-protocol 7(16): e2517. DOI: 10.21769/BioProtoc.2517.
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Ying Cao
Tongji University
Hi, Ruoning,
I'm wondering whether this protocol is only for live cells. Can I use this for fixed cells? Thanks!

Best,
Ying
2019/8/29 1:45:10 回复