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Dec 2017
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Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes
流式细胞术检测T淋巴细胞中LFA-1的再循环   

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Abstract

To enable cells to move forward, cell surface integrins are internalized into an endosomal compartment and subsequently intracellularly transported to be re-exposed at a new site on the cell membrane. Leukocytes are the fastest migrating cell type in the human body, which express the leukocyte-specific integrin LFA-1. Here, we describe a flow cytometry-based assay that allows the quantification of LFA-1 internalization and its re-expression on the cell surface in T lymphocytes. An advantage of using flow cytometry-based assay over biochemical methods is the low number of needed cells. This protocol can be also used to measure recycling of other receptors.

Keywords: Flow cytometry (流式细胞技术), Receptor (受体), LFA-1 (LFA-1), Endocytosis (内吞作用), Exocytosis (胞吐作用)

Background

Leukocytes need to be quick to extravasate from the vascular in order to defeat invading pathogens. To become effector cells, T lymphocytes need to migrate in the lymph nodes where they can encounter their specific antigen (Ley et al., 2007). LFA-1, which is the major integrin used by T lymphocytes to adhere and migrate, binds to its ligand intercellular adhesion molecule-1 (ICAM-1) on the endothelial or the antigen presenting cell (Evans et al., 2009). The reuse of LFA-1 is a dynamic process as it is internalized and intracellularly transported to be re-exposed to a new sight on the cell membrane for the cell to move forward (Svensson et al., 2012). The lysosomotropic amine Primaquine can be used to block intracellular transport and when used in T cells LFA-1 dependent migration is impaired (Stanley et al., 2012). The exact mechanism how LFA-1 is internalized and recycled isn’t fully understood. One method to investigate the internalization and re-exposure of LFA-1 in low number of cells is to label cells with the non-blocking antibody for LFA-1 (TS-2) and analyze the internalization and re-exposure of LFA-1 at different time points by flow cytometry (Samuelsson et al., 2017).

Materials and Reagents

  1. Plastic wares
    1. Pipette tips (2 μl, 20 μl, 200 μl and 1 ml)
    2. 96-well polystyrene round-bottom microwell plates (Thermo Fisher Scientific, Nunc, catalog number: 12-565-214)
    3. Microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001)
    4. 15 ml conical tubes (SARSTEDT, catalog number: 62.554.502)
  2. Cells (here we use human primary T lymphoblasts purified from whole blood. Other cell types can also be used)
  3. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7030)
  4. Primaquine diphosphate (PQ) (Sigma-Aldrich, catalog number: 160393)
  5. Paraformaldehyde, 16% w/v aq. soln., methanol free (VWR, catalog number: 43368.9M)
  6. Buffers
    1. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 14040133)
    2. Hanks balanced salt solution with or without Ca2+/Mg2+ (HBSS) (Thermo Fisher Scientific, Gibco, catalog numbers: 14025092 and 14175095)
    3. HEPES (Thermo Fisher Scientific, catalog number: 15630080)
  7. Antibodies
    1. LFA-1 antibody: Antibody purified from hybridoma TS2/4.1.1 (TS2/4.1.1, ATCC, catalog number: HB-244)
    2. Secondary Alexa Fluor® 647 donkey anti-mouse IgG (H+L) (Thermo Fisher, catalog number: A-31571)

Equipment

  1. Pipettes (0-2.5 μl, 2-20 μl, 20-200 μl, 100 μl-1 ml)
  2. Incubator
  3. Swing-out Centrifuge for microplates
  4. Flow cytometry (BD Biosciences, model: LSR II)

Software

  1. Flowjo, LLC (software for cytometry analysis)

Procedure

  1. Labeling of surface LFA-1 (see Figure 1 for an overview)
    1. Take 3.4 x 107 cells and wash in 15 ml ice cold HBSS wash buffer (Recipe 1). 
    2. Centrifuge at 300 x g for 10 min at 4 °C.
    3. Remove 1 x 106 cells for unstained control and fix these in 200 μl 3% PFA in migration buffer (Recipe 2) for 20 min on ice and wash in 1 ml ice cold migration buffer twice and resuspend in 200 μl ice cold 2% FBS in PBS and store until analysis at 4 °C.
    4. Resuspend 3.3 x 107 cells (Note 2) in 1 ml of ice cold 10 μg/ml TS2/4.1.1 antibody in migration buffer. 
    5. Incubate on ice for 30 min.
    6. Wash once with 10 ml ice cold migration buffer.
    7. Centrifuge at 300 x g for 10 min at 4 °C.
    8. Resuspend in 6 ml ice cold migration buffer and keep on ice until being used.


    Figure 1. An overview of the assay for recycling of LFA-1

  2. Labeling of intracellular LFA-1
    1. Take 1 ml of TS2-stained cells and divide into 3 microcentrifuge tubes (330 μl cells/tube) and add 0 μl from freshly made 30 mM PQ (Note 3) to one tube (control, 0 μM PQ) and 1.1 μl PQ to the second tube (100 μM PQ) and finally 3.3 μl PQ to the third tube (300 μM PQ).
    2. Take 110 μl into each well in a 96-well plate from each condition in Step B1 to make triplicates for time point 150 min and incubate for 30 min at 37 °C.
      Note: We repeat this procedure for the 120 min, 90 min, 60 min, 30 min, and 0 min time-points. Two minutes before each time point (Note 4), we prepare the PQ samples as described in Step B1. Precisely at the time points we take out the 96-well plate and add 110 μl into each well to make triplicates. The 96-well plate is returned to the 37 °C incubator after each step. 
    3. After final incubation take out the 96-well plate and put on ice and add the last triplicates (110 μl/well) for the 0 min time point.
    4. Add 100 μl of ice cold migration buffer into all wells and centrifuge the plate at 300 x g, 4 °C for 2 min.
    5. Resuspend cells in ice cold 2 μg/ml donkey anti-ms-AF647 in ice cold migration buffer with 0.1% BSA for 20 μl/sample.
    6. Incubate on ice for 20 min.
    7. Wash twice each with 200 μl ice cold migration buffer and centrifuge the plate at 300 x g, 4 °C for 2 min.
    8. Fix cell with 3% PFA in ice cold migration buffer for 20 min at 4 °C.
    9. Wash twice each with 200 μl ice cold migration buffer and centrifuge the plate at 300 x g, 4 °C for 2 min.
    10. Resuspend in 200 μl 2% FBS in PBS and transfer to FACS tube.
    11. Analyze samples using flow cytometry or store at 4 °C until analysis.

Data analysis

  1. For flow cytometry analysis, collect 30,000 gated events using FSC-A and SSC-A to avoid dead cells for each sample.
  2. Cells are then analyzed by using FSC-A and FSC-H to gate for single cells and then FL-4 to create histograms for the different time points and calculate Mean Fluorescence Intensity (MFI).
  3. To remove background from all samples subtract the MFI from unstained cells and calculate the mean of triplicates from each time point and condition.
  4. To calculate relative % of surface expression, we use the time point 0 min as 100%;

    Relative % of surface expression = Divide the mean of the given time point by the mean of time point 0.

    As integrins first internalize you will first see a reduction and then after some time a re-exposure of integrins on the surface. In the samples treated with PQ the re-exposure will be lower compared to non-treated cells (see Figure 2 for an example).


    Figure 2. An example of a result of LFA-1 recycling in presence and absence of PQ

Notes

  1. Take 6 x 105 cells per sample and use in triplicate for each time point. So for one condition with 6-time points, the total amount of the cells is 6 x 105 x 3 x 6 = 1.08 x 107 cells.
  2. Make a fresh 30 mM stock of Primaquine diphosphate (PQ) in PBS (= 13.86 mg/ml). 
  3. To prepare the next step, you need more or less 2 min to prepare. And you need to adjust this time to suit you.
  4. Here you can use a new 96-well plate for each time point and adjust time and use 2 plate centrifuges if you want to avoid taking out and in the plates from the incubator.

Recipes

  1. HBSS wash buffer
    HBSS without Ca2+/Mg2+
    10 mM HEPES
    Precool on ice before use
  2. Migration buffer
    HBSS with Ca2+/Mg2+ without phenol red (Gibco)
    5 mM Mg2+
    10 mM HEPES
    Precool on ice before use

Acknowledgments

The protocol was adapted from Samuelsson et al. (2017). This work was supported by Swedish Research Council awards K2010-80P-21592-01-4 and K2010-80X-215917-01-4, Foundation Olle Engquist Byggmästare, I&A Lundberg Research Foundation, Royal Swedish Academy of science, Royal Physiographic Society of Lund, Åke Wiberg, Jeanssons Foundation, Kocks Foundation, P&U Schybergs Foundation, Gyllenstiernska Krapperup Foundation, Gustav V 80 Jubilee Fund, Österlund Foundation, Nanna Svartz and Crafoord awards (to LS). Anna-Greta Crafoord postdoctoral fellowship and Royal Physiographic Society of Lund (KP) and Royal Physiographic Society of Lund (MS).

Competing interests

The authors declare no competing interest.

References

  1. Evans, R., Patzak, I., Svensson, L., De Filippo, K., Jones, K., McDowall, A. and Hogg, N. (2009). Integrins in immunity. J Cell Sci 122(Pt 2): 215-225.
  2. Ley, K., Laudanna, C., Cybulsky, M. I. and Nourshargh, S. (2007). Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7(9): 678-689.
  3. Samuelsson, M., Potrzebowska, K., Lehtonen, J., Beech, J. P., Skorova, E., Uronen-Hansson, H. and Svensson, L. (2017). RhoB controls the Rab11-mediated recycling and surface reappearance of LFA-1 in migrating T lymphocytes. Sci Signal 10(509) pii: eaai8629.
  4. Stanley, P., Tooze, S. and Hogg, N. (2012). A role for Rap2 in recycling the extended conformation of LFA-1 during T cell migration. Biol Open 1(11): 1161-1168.
  5. Svensson, L., Stanley, P., Willenbrock, F. and Hogg, N. (2012). The Galphaq/11 proteins contribute to T lymphocyte migration by promoting turnover of integrin LFA-1 through recycling. PLoS One 7(6): e38517.

简介

为了使细胞向前移动,将细胞表面整联蛋白内化到内体区室中,随后进行细胞内转运以在细胞膜上的新位点再次暴露。 白细胞是人体中最快的迁移细胞类型,其表达白细胞特异性整联蛋白LFA-1。 在这里,我们描述了基于流式细胞术的测定,其允许定量LFA-1内化及其在T淋巴细胞中的细胞表面上的重新表达。 使用基于流式细胞术的测定而不是生化方法的优点是所需细胞数量少。 该方案还可用于测量其他受体的再循环。

【背景】白细胞需要快速从血管中渗出以抵抗入侵的病原体。为了成为效应细胞,T淋巴细胞需要在淋巴结中迁移,在那里它们可以遇到它们的特异性抗原(Ley et al。>,2007)。 LFA-1是T淋巴细胞粘附和迁移的主要整合素,它与内皮细胞或抗原呈递细胞上的配体细胞间粘附分子-1(ICAM-1)结合(Evans 等。>,2009)。 LFA-1的再利用是一个动态过程,因为它被内化并在细胞内运输,再次暴露于细胞膜上的新视野,使细胞向前移动(Svensson et al。>,2012 )。溶酶体丙胺Primaquine可用于阻断细胞内转运,当用于T细胞时,LFA-1依赖性迁移受损(Stanley et al。>,2012)。 LFA-1如何内化和回收的确切机制尚不完全清楚。研究LFA-1在少量细胞中的内化和再暴露的一种方法是用LFA-1(TS-2)的非阻断抗体标记细胞并分析LFA-1的内化和再暴露。在不同的时间点通过流式细胞术(Samuelsson et al。>,2017)。

关键字:流式细胞技术, 受体, LFA-1, 内吞作用, 胞吐作用

材料和试剂

  1. 塑料制品
    1. 移液器吸头(2μl,20μl,200μl和1 ml)
    2. 96孔聚苯乙烯圆底微孔板(Thermo Fisher Scientific,Nunc,目录号:12-565-214)
    3. 微量离心管(SARSTEDT,目录号:72.690.001)
    4. 15毫升锥形管(SARSTEDT,目录号:62.554.502)
  2. 细胞(这里我们使用从全血中纯化的人类原发性T淋巴母细胞。也可以使用其他细胞类型)
  3. 牛血清白蛋白(BSA)(西格玛奥德里奇,目录号:A7030)
  4. Primaquine diphosphate(PQ)(西格玛奥德里奇,目录号:160393)
  5. 多聚甲醛,16%w / v水溶液。溶剂,不含甲醇(VWR,目录号:43368.9M)
  6. 缓冲区
    1. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,目录号:14040133)
    2. Hanks平衡盐溶液,有或没有Ca 2 + / Mg 2 + (HBSS)(Thermo Fisher Scientific,Gibco,目录号:14025092和14175095)
    3. HEPES(赛默飞世尔科技,目录号:15630080)
  7. 抗体
    1. LFA-1抗体:从hybridoma TS2 / 4.1.1(TS2 / 4.1.1,ATCC,目录号:HB-244)纯化的抗体
    2. 次级Alexa Fluor ® 647驴抗小鼠IgG(H + L)(Thermo Fisher,目录号:A-31571)

设备

  1. 移液器(0-2.5μl,2-20μl,20-200μl,100μl-1 ml)
  2. 恒温箱
  3. 摆动式离心机用于微孔板
  4. 流式细胞仪(BD Biosciences,型号:LSR II)

软件

  1. Flowjo,LLC(用于细胞计数分析的软件)

程序

  1. 表面LFA-1的标签(概述见图1)
    1. 取3.4×10 7 细胞,用15ml冰冷的HBSS洗涤缓冲液(配方1)洗涤。 
    2. 在4℃下以300 x g >离心10分钟。
    3. 去除1×10 6 细胞用于未染色的对照,并将其在200μl3%PFA的迁移缓冲液(配方2)中在冰上固定20分钟,并在1ml冰冷的迁移缓冲液中洗涤两次并重悬于在PBS中加入200μl冰冷的2%FBS并储存直至在4℃下分析。
    4. 将3.3×10 7 细胞(注释2)重悬于迁移缓冲液中的1ml冰冷的10μg/ ml TS2 / 4.1.1抗体中。 
    5. 在冰上孵育30分钟。
    6. 用10ml冰冷的迁移缓冲液洗涤一次。
    7. 在4℃下以300 x g >离心10分钟。
    8. 重悬于6毫升冰冷的迁移缓冲液中并保持在冰上直至使用。


    图1. LFA-1回收分析的概述

  2. 标记细胞内LFA-1
    1. 取1 ml TS2染色细胞,分成3个微量离心管(330μl细胞/管),加入0μl新鲜制备的30 mM PQ(注3)至一管(对照,0μMPQ)和1.1μlPQ至第二管(100μMPQ),最后3.3μlPQ到第三管(300μMPQ)。
    2. 从步骤B1中的每个条件中取110μl进入96孔板的每个孔中,使时间点重复150分钟,并在37℃下孵育30分钟。
      注意:我们重复此程序120分钟,90分钟,60分钟,30分钟和0分钟的时间点。在每个时间点前两分钟(注4),我们按步骤B1所述准备PQ样品。准确地说,在时间点我们取出96孔板并在每个孔中加入110μl进行三次重复。每步完成后,将96孔板放回37°C培养箱中。>
    3. 在最后孵育后取出96孔板并置于冰上并在0分钟时间点加入最后一式三份(110μl/孔)。
    4. 向所有孔中加入100μl冰冷的迁移缓冲液,并在300℃下将板离心4℃,2分钟。
    5. 将细胞重悬于冰冷的2μg/ ml驴抗-ms-AF647冰冷迁移缓冲液中,含0.1%BSA,20μl/样品。
    6. 在冰上孵育20分钟。
    7. 用200μl冰冷的迁移缓冲液洗涤两次,并将板在300℃下离心4℃,4℃离心2分钟。
    8. 将具有3%PFA的细胞在冰冷的迁移缓冲液中在4℃下固定20分钟。
    9. 用200μl冰冷的迁移缓冲液洗涤两次,并将板在300℃下离心4℃,4℃离心2分钟。
    10. 重悬于PBS中的200μl2%FBS并转移至FACS管。
    11. 使用流式细胞仪分析样品或在4°C储存直至分析。

数据分析

  1. 对于流式细胞术分析,使用FSC-A和SSC-A收集30,000个门控事件,以避免每个样品的死细胞。
  2. 然后通过使用FSC-A和FSC-H来分析细胞以对单个细胞进行门控,然后使用FL-4来分析不同时间点的直方图并计算平均荧光强度(MFI)。
  3. 要从所有样品中去除背景,从未染色的细胞中减去MFI,并从每个时间点和条件计算一式三份的平均值。
  4. 为了计算表面表达的相对%,我们使用0分钟的时间点作为100%;

    表面表达式的相对百分比=将给定时间点的平均值除以时间点0的平均值。

    当整合素首先内化时,您将首先看到减少,然后在一段时间后重新暴露表面上的整合素。在用PQ处理的样品中,与未处理的细胞相比,再次暴露将更低(例如,参见图2)。


    图2.在存在和不存在PQ的情况下回收LFA-1的结果示例

笔记

  1. 每个样品取6×10个 5 细胞,每个时间点一式三份使用。因此,对于具有6个时间点的一个条件,细胞的总量是6×10 5个/ 5×10×10×1.08×10 10个细胞。
  2. 在PBS(= 13.86 mg / ml)中制备新鲜的30 mM Primaquine diphosphate(PQ)原液。 
  3. 要准备下一步,您需要或多或少2分钟的准备时间。你需要调整这个时间以适合你。
  4. 在这里,您可以为每个时间点使用新的96孔板,并调整时间并使用2板离心机,如果您想避免从培养箱中取出并进入板中。

食谱

  1. HBSS洗涤缓冲液
    没有Ca 2 + / Mg 2 + 的HBSS
    10 mM HEPES
    使用前在冰上预冷
  2. 迁移缓冲区
    没有酚红(Gibco)的Ca 2 + / Mg 2 + 的HBSS
    5mM Mg 2 +
    10 mM HEPES
    使用前在冰上预冷

致谢

该协议改编自Samuelsson 等人>(2017)。这项工作得到瑞典研究委员会奖项K2010-80P-21592-01-4和K2010-80X-215917-01-4,基金会OlleEngquistByggmästare,I& A Lundberg研究基金会,瑞典皇家科学院,皇家地理学会的支持。 Lund,ÅkeWiberg,Jeanssons Foundation,Kocks Foundation,P& U Schybergs Foundation,Gyllenstiernska Krapperup Foundation,Gustav V 80 Jubilee Fund,ÖsterlundFoundation,Nanna Svartz和Crafoord奖(LS)。 Anna-Greta Crafoord博士后奖学金和隆德皇家地理学会(KP)和隆德皇家地理学会(MS)。

利益争夺

作者声明没有竞争利益。

参考

  1. Evans,R.,Patzak,I.,Svensson,L.,De Filippo,K.,Jones,K.,McDowall,A。和Hogg,N。(2009)。 整合免疫力。 J Cell Sci > 122(Pt 2):215-225。
  2. Ley,K.,Laudanna,C.,Cybulsky,M。I.和Nourshargh,S。(2007)。 进入炎症部位:更新了白细胞粘附级联。 Nat Rev Immunol > 7(9):678-689。
  3. Samuelsson,M.,Potrzebowska,K.,Lehtonen,J.,Beech,J.P.,Skorova,E.,Uronen-Hansson,H。和Svensson,L。(2017)。 RhoB控制Rab11介导的LFA-1在迁移的T淋巴细胞中的再循环和表面再现。 科学信号> 10(509)pii:eaai8629。
  4. Stanley,P.,Tooze,S。和Hogg,N。(2012)。 Rap2在T细胞迁移过程中回收LFA-1扩展构象的作用。 Biol Open > 1(11):1161-1168。
  5. Svensson,L.,Stanley,P.,Willenbrock,F。和Hogg,N。(2012)。 Galphaq / 11蛋白通过循环促进整合素LFA-1的转换,促进T淋巴细胞迁移。 PLoS One > 7(6):e38517。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Potrzebowska, K., Lehtonen, J., Samuelsson, M. and Svensson, L. (2018). Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes. Bio-protocol 8(23): e3104. DOI: 10.21769/BioProtoc.3104.
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