Name: molecular diffusion in plasma membranes of primary lymphocytes measured by fluorescence correlation spectroscopy
Uploaded: 2017-02-01T12:30:00
Description: Watch this Scientific Journal Video about Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy at JoVE.com

我们感谢Vladana武克杰维奇博士，分子医学中心，卡罗林斯卡学院对维护蔡司Confocor 3仪器，并为有关小区测量有用的技巧。这项研究是由来自Vetenskapsrådet资助（批准号2012- 1629），马格努斯Bergvalls stiftelse，并从Stiftelsen克拉斯Groschinskys minnesfond资助。

1.染色的FCS <ol><li>隔离用磁珠标记，按照生产商的方案16脾淋巴细胞的小鼠NK细胞。使用每个样品2-3×10 5个细胞用于以下的步骤。 注意：使用阴性选择，以丰富的靶细胞群体，留下不变，从而使细胞可以单独使用初级抗体进行标记。请参阅从小鼠2细胞分离更详细的信息参考2。 </li><li>用于表达Fc受体鼠细胞，阻断Fc受体与抗体克隆...

<ol>
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J., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy+for+the+study+of+membrane+dynamics+and+protein/lipid+interactions.">Fluorescence correlation spectroscopy for the study of membrane dynamics and protein/lipid interactions.</a> Methods. 46, 116-122 (2008).</li><li>Machan, R., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+applications+of+fluorescence+correlation+spectroscopy+in+live+systems.">Recent applications of fluorescence correlation spectroscopy in live systems.</a> FEBS Lett. 588, 3571-3584 (2014).</li><li>Mutze, J., Ohrt, T., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy+in+vivo.">Fluorescence correlation spectroscopy in vivo.</a> Laser Photon Rev. 5, 52-67 (2011).</li><li>Lidke, D. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+correlation+spectroscopy:+principles+and+applications.">Image correlation spectroscopy: principles and applications.</a> Cold Spring Harb Protoc. , 336-348 (2015).</li><li>Sankaran, J., Shi, X., Ho, L. Y., Stelzer, E. H., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImFCS:+a+software+for+imaging+FCS+data+analysis+and+visualization.">ImFCS: a software for imaging FCS data analysis and visualization.</a> Opt Express. 18, 25468-25481 (2010).</li><li>Liu, P., Ahmed, S., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F-techniques:+advances+in+receptor+protein+studies.">The F-techniques: advances in receptor protein studies.</a> Trends Endocrinol Metab. 19, 181-190 (2008).</li><li>Manley, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+mapping+of+single-molecule+trajectories+with+photoactivated+localization+microscopy.">High-density mapping of single-molecule trajectories with photoactivated localization microscopy.</a> Nat Methods. 5, 155-157 (2008).</li><li>Bacia, K., Haustein, E., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy:+principles+and+applications.">Fluorescence correlation spectroscopy: principles and applications.</a> Cold Spring Harb Protoc. , 709-725 (2014).</li><li>Haustein, E., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy:+novel+variations+of+an+established+technique.">Fluorescence correlation spectroscopy: novel variations of an established technique.</a> Annu Rev Biophys Biomol Struct. 36, 151-169 (2007).</li><li>Widengren, J., Thyberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FCS+cell+surface+measurements--photophysical+limitations+and+consequences+on+molecular+ensembles+with+heterogenic+mobilities.">FCS cell surface measurements--photophysical limitations and consequences on molecular ensembles with heterogenic mobilities.</a> Cytometry A. 68, 101-112 (2005).</li><li>North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing?+A+beginners'+guide+to+practical+pitfalls+in+image+acquisition.">Seeing is believing? A beginners' guide to practical pitfalls in image acquisition.</a> J Cell Biol. 172, 9-18 (2006).</li><li>Meinhardt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+NK+cell+magnetic+bead+isolation+methods+on+phenotype+and+function+of+murine+NK+cells.">Influence of NK cell magnetic bead isolation methods on phenotype and function of murine NK cells.</a> J Immunol Methods. 378, 1-10 (2012).</li><li>Bacia, K., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+guidelines+for+dual-color+fluorescence+cross-correlation+spectroscopy.">Practical guidelines for dual-color fluorescence cross-correlation spectroscopy.</a> Nat Protoc. 2, 2842-2856 (2007).</li><li>Gendron, P. O., Avaltroni, F., Wilkinson, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+coefficients+of+several+rhodamine+derivatives+as+determined+by+pulsed+field+gradient-nuclear+magnetic+resonance+and+fluorescence+correlation+spectroscopy.">Diffusion coefficients of several rhodamine derivatives as determined by pulsed field gradient-nuclear magnetic resonance and fluorescence correlation spectroscopy.</a> J Fluoresc. 18, 1093-1101 (2008).</li><li>Petrasek, Z., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+measurement+of+diffusion+coefficients+using+scanning+fluorescence+correlation+spectroscopy.">Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy.</a> Biophys J. 94, 1437-1448 (2008).</li><li>Stromqvist, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+FCCS+procedure+applied+to+Ly49A-MHC+class+I+cis-interaction+studies+in+cell+membranes.">A modified FCCS procedure applied to Ly49A-MHC class I cis-interaction studies in cell membranes.</a> Biophys J. 101, 1257-1269 (2011).</li><li>Widengren, J., Mets, U., Rigler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Correlation+Spectroscopy+of+Triplet-States+in+Solution+-+a+Theoretical+and+Experimental-Study.">Fluorescence Correlation Spectroscopy of Triplet-States in Solution - a Theoretical and Experimental-Study.</a> J Phys Chem. 99, 13368-13379 (1995).</li><li>Humpolickova, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+diffusion+laws+within+cellular+membranes+by+Z-scan+fluorescence+correlation+spectroscopy.">Probing diffusion laws within cellular membranes by Z-scan fluorescence correlation spectroscopy.</a> Biophys J. 91, L23-L25 (2006).</li><li>Guo, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+approach+to+the+analysis+of+fluorescence+correlation+spectroscopy+data+II:+application+to+simulated+and+in+vitro+data.">Bayesian approach to the analysis of fluorescence correlation spectroscopy data II: application to simulated and in vitro data.</a> Anal Chem. 84, 3880-3888 (2012).</li><li>Ries, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+suppression+of+sample-related+artifacts+in+Fluorescence+Correlation+Spectroscopy.">Automated suppression of sample-related artifacts in Fluorescence Correlation Spectroscopy.</a> Opt Express. 18, 11073-11082 (2010).</li><li>Chiu, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+kinetics+of+the+androgen+receptor+shown+by+multimodal+Image+Correlation+Spectroscopy+(mICS).">Intracellular kinetics of the androgen receptor shown by multimodal Image Correlation Spectroscopy (mICS).</a> Sci Rep. 6, 22435 (2016).</li><li>Wegel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cellular+structures+in+super-resolution+with+SIM,+STED+and+Localisation+Microscopy:+A+practical+comparison.">Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.</a> Sci Rep. 6, 27290 (2016).</li></ol>

这个协议的FCS可用于表面分子上的所有类型的免疫细胞（鼠的，人的，或其他物种）的分子动力学的评估。 FCS措施时空分子动力学下降到活细胞中的单分子的分辨率。分子密度，以及感兴趣的蛋白质的扩散速度和集群动力学，可以从自相关曲线中提取。 荧光标记是成功的FCS实验枢轴重要性。的细胞膜上的感兴趣的蛋白质传统上一直使用抗体标记和抗体常常最方便对幼稚免疫?...

许多免疫细胞的功能依赖于分子扩散和薄膜内的相互作用。生物膜是复杂的，许多因素可能对免疫细胞的功能，可以细胞膜1内影响蛋白的平移扩散的速度很重要。我们最近发现，属于先天性免疫系统天然杀伤（NK）细胞，淋巴细胞，在细胞膜呈现2研究蛋白质的差扩散取决于NK细胞活化2的状态。 荧光相关光谱（FCS）是一种技术，其能够生物膜内量化分子扩散率。它报告的平均扩散速度的荧光标记的分子在一个固定的体积，共焦显微镜的通常的焦点。它是基于波动的荧光时在分子的运动所发生的测量在稳定状态下的系统。 FCS已广泛用于研究荧光染料和蛋白质的扩散，无论是在溶液和脂质膜之内。影响扩散速率其他输出参数也可以用这种方式来间接地研究（ 例如，蛋白质或细胞膜上的分子相互作用的构象变化）3，4。 FCS的突出相比，由于其高灵敏度的其他技术，允许单分子检测的可能性。它可以很好地用于分子浓度在纳摩尔到毫摩尔范围内，这是典型的大多数蛋白质5的内源表达水平。另外，FCS可以给所研究的容积内的蛋白质的绝对数量的近似，而大多数其它技术仅得到约蛋白质表达水平的相对信息。其他的方法来测量分子扩散率中膜包括FLUO漂白（FRAP）后rescence恢复，单粒子跟踪（SPT），多个针孔FCS和图像相关方法。 FRAP和图像相关方法集合技术，其通常不提供有关分子10的绝对数量的信息。相对于SPT，FCS的吞吐量是关于表征人口平均水平。由于激光焦点中存在的分子的平均扩散速度被测量，而不是单分子速度的分析也是要求不高。此外，除非专门显微镜可用11，SPT不能给关于浓度的任何信息，因为标准的SPT标签必须非常低，以允许单个分子的鉴定。另一方面，FCS要求所研究的分子是移动的。它根本不会发现任何假定不动的分数或分子移动的速度很慢。分子的扩散速率居住超过约十分之一的采集时间不会FCS测量3，12代表正确的时间越长，内对焦。因此，通过FCS记录扩散系数往往比从像FRAP和SPT，其中接近对不动和非常慢的部分都考虑在内，以及技术报告的扩散速率快。 SPT也会给分子群体比FCS将内扩散速率的可变性的一个更详细的说明。 FCS的量化的荧光强度随时间的兴奋体积内的波动。在膜的测量的情况下，这转化为在膜的照明面积。在本文中，我们利用的事实，这样的波动是由表现出布朗扩散分子诱导和中和了的激发体积因而移动。也有用于fluctua几个其它可能的来源系统蒸发散在荧光信号，如闪烁的或三重态的荧光团，环境效应的存在，结合-解除绑定的配位体，或整个细胞膜的运动。这些推定的误差源需要以准确解释结果12,13设计一个FCS的实验时，必须考虑到。通常，在生物膜的横向扩散速率是由于拥挤和相互作用低，两膜蛋白之间和蛋白质和细胞骨架之间。从历史上看，在膜中使用的FCS就此受到阻碍缺乏光稳定的荧光团，它被要求，以避免在通过激励焦点14延长运输时间漂白。然而，今天，有很多合适的光稳定的染料选择。在探测器和其他硬件显著的改进也让荧光prote检测插件和低亮度的染料。这里，对于FCS的使用鼠初级淋巴细胞中的应用，其中，感兴趣的蛋白质标记有荧光标记的抗体的基本协议，进行说明。适合以提取扩散系数和分子密度的自相关曲线的方法还示出。该协议的目的是容易地随后免疫学家与技术来研究分子的扩散没有以往的经验。然而，激光共聚焦显微镜的一个基本的了解，预计（获得这一基本认识，见参考文献15）。这个协议可以相对容易地适应其他悬浮的细胞，这两种细胞系和原代细胞。有经验的FCS的用户，更精致的分析方法存在，其中一些在讨论中描述。

<table border="0" cellpadding="0" cellspacing="0" width="1206">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:321px;">MACS NK Cell Isolation Kit mouse II</td>
			<td style="width:367px;">Miltenyi Biotec NordenAB</td>
			<td style="width:181px;">130-096-892</td>
			<td style="width:337px;">Negative selection of NK cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Fetal Bovine Serum</td>
			<td style="width:367px;">Sigma-Adlrich</td>
			<td style="width:181px;">F7524</td>
			<td style="width:337px;">Heat inactivated</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Phosphate Buffered Saline</td>
			<td style="width:367px;">-</td>
			<td style="width:181px;">-</td>
			<td style="width:337px;">Made in house&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Roswell Park Memorial Institute medium 1640</td>
			<td style="width:367px;">PAA The Cell Culture Company&#160;</td>
			<td style="width:181px;">E15-848</td>
			<td style="width:337px;">Transparent medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Antibody clone 2.4G2</td>
			<td style="width:367px;">Thermo Fischer Scientific</td>
			<td style="width:181px;">553140</td>
			<td style="width:337px;">For blocking Fc-receptors.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-Ly49A antibody&#160;</td>
			<td rowspan="2" style="width:367px;"></td>
			<td rowspan="2" style="width:181px;"></td>
			<td rowspan="2" style="width:337px;">Monoclonal antibody made in house and conjugated in house to Alexa fluor 647</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Clone JR9.318</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-H-2Dd antibody&#160;</td>
			<td rowspan="2" style="width:367px;">BD Pharmingen</td>
			<td rowspan="2" style="width:181px;">558915</td>
			<td rowspan="2" style="width:337px;">Conjugated in house to MFP488</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Clone 34.5.8S</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">MFP488</td>
			<td style="width:367px;">Mobiotech</td>
			<td style="width:181px;">MFP-A2181</td>
			<td style="width:337px;">Fluorescent dye for antibody conjugation.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Poly-L-Lysine</td>
			<td style="width:367px;">Sigma-Aldrich</td>
			<td style="width:181px;">P8920</td>
			<td style="width:337px;">Diluted in distilled water (1.10)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:321px;">Poly-L-Lysine (20 kDa) grafted with polyethylene glycol (2 kDa)</td>
			<td style="width:367px;">SuSoS AG</td>
			<td style="width:181px;">PLL(20)-g[3.5]-PEG(2)</td>
			<td style="width:337px;">Diluted in PBS (pH 7.4) to 0.5 mg/ml.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:321px;">Rhodamine 110 chloride</td>
			<td style="width:367px;">Sigma-Aldrich</td>
			<td style="width:181px;">432202</td>
			<td style="width:337px;">Known diffusion coefficient: 3.3 &#215; 10&#8722;10 m2/s 19</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:321px;">Alexa fluor 647</td>
			<td style="width:367px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:181px;">A20006</td>
			<td style="width:337px;">Known diffusion coefficient: 4.4 &#215; 10&#8722;10 m2/s 20</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Confocal microscope&#160;</td>
			<td style="width:367px;">Zeiss</td>
			<td style="width:181px;">LSM510</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Software: Confocor 3</td>
			<td style="width:367px;">Zeiss</td>
			<td style="width:181px;"></td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Software: Matlab with curve fitting toolbox</td>
			<td style="width:367px;">Matlab</td>
			<td style="width:181px;">Version R2013b</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Nunc Lab-Tek Chambered Coverglass</td>
			<td style="width:367px;">Thermo-scientific&#160;</td>
			<td style="width:181px;">155411</td>
			<td style="width:337px;">8 wells, 1.0 borosilicate bottom</td>
		</tr>
	</tbody>
</table>

molecular diffusion in plasma membranes of primary lymphocytes measured by fluorescence correlation spectroscopy

荧光相关光谱（FCS）是用于与高空间和时间分辨率研究生物膜内分子的扩散的强大技术。 FCS可以量化荧光标记的分子的分子浓度和扩散系数在细胞膜。该技术具有探索稳态免疫细胞的细胞膜分子的分子扩散特性（ 即，在不影响在实际测量时间的结果过程）的能力。 FCS是适合研究那些在典型的最内源性蛋白的水平表达的蛋白质的扩散。在此，展示了一个简单的和可靠的方法来确定主淋巴细胞细胞膜蛋白质的扩散率。以执行对抗体染色的活细胞和采集后通常发生的观测的测量的有效方法进行说明。最近进展在光稳定的荧光染料的开发可以通过缀合的感兴趣的抗体拨出不测量期间广泛漂白染料被利用。另外，这允许检测慢慢扩散实体，这是在细胞膜中表达的蛋白质的一个共同特点。该分析程序，从生成的自相关曲线中提取浓度的分子和扩散的参数被突出显示。总之，对于FCS测量一个基本协议被提供;它可以跟随免疫学家以共聚焦显微镜的理解，但是有用于测量动态参数，如分子的扩散速率的技术没有其他以往的经验。

一个典型的结果将产生在10毫秒到400毫秒膜蛋白范围内的运输时间自相关曲线。分子的数目可以为内源性表达的蛋白质0.5之间变化到每平方微米约200。仔细检查CPM高于预期不低。这可能意味着有背景信号的影响。作为一个经验法则，即接受的分析对细胞的CPM信号不应在同一激光功率自由扩散抗体是CPM低于33％。植物检疫措施应与激光功率线性扩展。用于初级免疫的?...

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Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

由荧光相关光谱测量的一次淋巴细胞的细胞膜分子扩散

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

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