We thank Dr. Vladana Vukojevi&#231;, Center for Molecular Medicine, Karolinska Institutet for the maintenance of the Zeiss Confocor 3 instrument and for helpful tips regarding cell measurements.&#160;This study was funded by grants from Vetenskapsr&#229;det (grant number 2012- 1629), Magnus Bergvalls stiftelse, and from Stiftelsen Claes Groschinskys minnesfond.

1. Staining for FCS
<ol>
	<li>Isolate murine NK cells from spleen lymphocytes using magnetic bead labelling, as per the manufacturer&#39;s protocol16. Use 2-3 x 105 cells per sample for the following steps. 
	NOTE: Use negative selection to enrich the target cell population, leaving it untouched, so that the cells can be labelled using primary antibodies alone. See reference2 for more detailed information on cell isolation from mice2</sup...

<ol>
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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. 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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>

The authors have nothing to disclose.

This protocol for FCS can be used for the assessment of the molecular dynamics of surface molecules on all types of immune cells (murine, human, or other species). FCS measures spatio-temporal molecular dynamics down to single-molecule resolution in live cells. The molecular density, as well as the diffusion rate and clustering dynamics of the proteins of interest, can be extracted from the autocorrelation curves.
The fluorescent labeling is of pivotal importance for successful FCS experiments...

Many immune cells functions rely on molecular diffusion and interactions within membranes. Biological membranes are complex, and many factors that may be important for the function of immune cells can influence the speed of translational diffusion of proteins within cellular membranes1. We recently showed that natural killer (NK) cells, lymphocytes belonging to the innate immune system, exhibit differential diffusion of two studied proteins at the cell membrane depending on the state of NK cell activation2.
Fluorescence correlation spectroscopy (FCS) is a technique that is capable of quantifying molecular diffusion rates within biological membranes. It reports the average diffusion rate of fluorescently labeled molecules in a fixed volume, typically the focus of a confocal microscope. It is based on the measurement of the fluctuations in fluorescence that occur upon molecular movement in a system in steady state. FCS has been widely used for studying the diffusion of fluorescent dyes and proteins, both in solution and within lipid membranes. Other output parameters affecting the diffusion rate can also be indirectly studied in this fashion (e.g., conformational changes of proteins or interactions of molecules on cell membranes)3,4. FCS stands out compared to other techniques due to its high sensitivity, allowing the possibility for single-molecule detection. It works well for molecular concentrations in the nanomolar to millimolar range, which is typical for endogenous expression levels of most proteins5. Furthermore, FCS can give an approximation of the absolute number of proteins within the studied volume, while most other techniques only give relative information about protein expression levels. Other methods to measure molecular diffusion rates within membranes include fluorescence recovery after photobleaching (FRAP), single particle tracking (SPT), multiple pinhole FCS, and image correlation methods. FRAP and image correlation methods are ensemble techniques, which generally do not give information about the absolute number of molecules10. Compared to SPT, the throughput of FCS is higher in regard to characterizing the population average. The analysis is also less demanding since the average diffusion rate of the molecules present within the laser focus is measured, rather than the rate of single molecules. Also, unless specialized microscopes are available11, SPT cannot give any information about concentrations, since standard SPT labeling must be very low to allow for the identification of single molecules. On the other hand, FCS requires the molecules under study to be mobile. It will simply not detect any putative immobile fractions or molecules moving very slowly. The diffusion rate of molecules that reside within the focus longer than approximately one tenth of the acquisition time will not be correctly represented in FCS measurements3,12. Therefore, diffusion coefficients recorded by FCS tend to be faster than diffusion rates reported from techniques like FRAP and SPT, where the close-to-immobile and very slow fractions are taken into account as well. SPT will also give a more detailed description of the variability of diffusion rates within the molecular population than FCS will.
FCS quantifies the fluctuation of fluorescence intensity over time within the excited volume. In the case of membrane measurements, this translates to the illuminated area of the membrane. In this paper, we utilize the fact that such fluctuations are induced by molecules exhibiting Brownian diffusion and are thus moving in and out of the excitation volume. There are also several other possible sources for the fluctuations in the fluorescence signal, such as blinking or the presence of a triplet state in the fluorophores, environmental effects, binding-unbinding of the ligand, or movement of the entire cell membrane. These putative error sources need to be taken into consideration when designing an FCS experiment in order to accurately interpret the results12,13. Typically, lateral diffusion rates in biological membranes are low due to crowding and interactions, both between membrane proteins and between proteins and the cytoskeleton. Historically, the use of FCS in membranes has thus been hampered by the lack of photo-stable fluorophores, which are required to avoid bleaching during the extended transit times through the excitation focus14. However, today, there are plenty of options for suitable photo-stable dyes. Significant improvements in detectors and other hardware also allow the detection of fluorescent proteins and dyes of lower brightness. Here, a basic protocol for the application of FCS using murine primary lymphocytes, where the protein of interest is labeled with a fluorescently tagged antibody, is described. An approach to fit the autocorrelation curves in order to extract the diffusion coefficient and the molecular density is also shown. The protocol aims at being easily followed by immunologists with no previous experience of techniques to study the diffusion of molecules. However, a basic understanding of confocal microscopy is expected (to gain this basic understanding, see reference15). This protocol can relatively easily be adapted to other suspension cells, both cell lines and primary cells. For more experienced FCS users, more refined analysis methods exist, some of which are described in the discussion.

<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

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A typical result will generate an autocorrelation curve with a transit time in the range of 10 msec to 400 msec for membrane proteins. The number of molecules can vary between 0.5 to around 200 per &#956;m2 for endogenously expressed proteins. Check carefully that the CPM is not lower than expected. This may mean that there is an influence of the background signal. As a rule of thumb, the CPM signal on cells that is accepted for analysis should not be lower than 33% of the CPM ...

Watch this Scientific Journal Video about Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy at JoVE.com

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.

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

The overall goal of this protocol is to measure the diffusion rate of a labeled protein in membranes of primary immune cells using fluorescence correlation spectroscopy. This method can help identify key questions in the immunology and cell biology fields, such as identifying natural killer cells with a more active membrane receptor dynamics. The main advantage of this technique is that it is used with live cells, so it accurately reflects the natural state on how molecules move.The implications of this technique extend towards immunotherapy of cancer if different natural killer cell traits, such as molecular diffusion, are linked to their function that efficiency can be predicted better. Though this method can provide insight into molecular movement in immune cells, it can also be applied to other primary cells and cell lines in any discipline. Open the software in expert mode, with the option Scan New Images selected.Select the 40X water objective lens on the confocal laser scanning microscope. Under the Acquire tab, press the Config and Scan buttons to open the Configuration Control and the Scan Control. Under the ConfoCor tab, press Measure to open the Measurement window.Next, create a folder to save the FCS measurements. Start a new image database by clicking New under the File tab. Turn on the halogen lamp.Using the Laser button, turn on the relevant lasers for exciting the fluorophores. Then specify the settings for image capture in the Configuration Control window. This includes the excitation laser, excitation and emission filters, light path, and detectors in use.Similarly, go to the Measurement window and adjust the FCS settings under the System Configuration tab. Activate the detection channels for the FCS recordings by toggling Ch 1 for the green channel, and Ch 2 for the red channel. While the laser stabilizes, which may take up to 30 minutes, adjust the pinhole.First, place a 50 microliter drop of dye centrally into one well of an eight-well chambered coverglass slide coated with poly-L-lysine. The adjacent wells are used to make cell measurements as the calibration is slide-dependent. Put a drop of distilled water on the 40X water objective lens and position the slide.Then press Vis to select the visible light source and focus on the central position of the fluorophore droplet. In the software Measurement window, select the Acquisition tab and under Positions, toggle the current position. Then return to the System Configuration tab in the same window, and set a high or saturating power for the green laser.To test the stability of the signal, press Start to open a new window displaying the Time trace and Auto correlation curve. Check that the fluorescent signal is high, stable, and that the y axis shows fluctuation in the kilohertz range. Lower values could be due to imprecise focusing or dye degradation.Next, in the Measurement window, select the O Adjust button. Then select the correct detection channel and press Auto Adjust X.If the upper limit of the resulting curve is missing or not centered, mark the Coarse option and press Auto Adjust X again. After adjusting the X direction, do the same for the Y direction by pressing on Auto Adjust Y.Deselect Coarse and alternate between adjusting X and Y by pressing Auto Adjust until both have clear maxima and stable values.If the cells labeled by two different fluorophores, add a drop of red fluorophore solution to an adjacent chamber. Then calibrate the second channel. Set the red laser to a high power, and repeat the stability calibration procedure.Next, we measure the transit time of freely-diffusing fluorophores through the focus. And we use the stage then to calculate the area of the cell membrane that is within the focus. To start, focus on a droplet of solution by carefully carrying out the adjustment procedure.Then go to the Acquisition tab in the Measurement window and set the acquisition time to 30 seconds with three repeats. Set the laser power at near saturating and press Start to take a measurement. Record the counts per molecule or CPM after the measurement is complete.Then take new measurements making iterative reductions to the laser power. Within this power range, include the expected power of the forthcoming cell measurements. Next check that the CPM values scale linearly with the laser power.Provided the rest of the scale is linear, saturation at the highest power is acceptable. The first time a newly-conjugated fluorescent antibody is used, perform an FCS measurement of freely-diffusing antibodies to quantify the expected CPM. And it is important to use wells coated with PEG Poly-L-Lysine for free antibodies.For this procedure, coat a chambered slide with 200 microliters of Poly-L-Lysine grafted to polyethylene glycol. Let the solution adhere for an hour at room temperature. The stock solution can be refrigerated for up to two weeks.And the powder stock should be stored in the freezer. Later, remove the liquid and allow the chamber to air dry. At the microscope, dilute the antibody in PBS to between one and 10 micrograms per milliliter.Proceed to measure the transit time of free antibodies as described in the previous section for free fluorophores. To take measurement in a cell, add 125 microliters of cells in PBS plus 1%FCS from the antibody-labeled cell suspension. And 150 microliters of transparent Roswell Park Memorial Institute Medium 1640 into an empty well in the PLL-coated glass slide.Let the cells incubate in the dark at the measurement temperature for at least 20 minutes before proceeding, so that the cells can attach and equilibrate. Start the imaging with fast XY scans to find the lowest possible laser power needed to view the cells. Then center the image on a round cell of average brightness where the membrane is clearly defined.It is crucial that there is no fluorescent signal in the cytoplasm. Zoom in until the cell covers most of the image. Then focus on the top of the cell membrane.Start at the center of the cell and move upwards until the center of the image shows a small round area with even fluorescence. Careful placement of the focus is crucial. If the focus is not in the membrane, the fluorescence detected will not come from the fluorescently-labeled membrane receptors.And thus the results will not be reflective of their behavior. Now under the Acquisition tab of the Measurement window, select a position for FCS measurement by activating the Crosshair. Then set the number of repeats to between six and 10 and set the Measure Time to 10 seconds per repeat.Now return to the System Configuration tab of the Measurement window and lower the laser power for FCS measurements to between one and 10 microwatts. Then press Start. If the intensity trace drops by more than 30%during the first 10 seconds, the cell has then been bleached too much and another cell must be measured at a lower power.If the signal is lost, adjust the focus in the Z direction and restart the measurement. After the measurement is complete look in the Scan Control window to check that the cell is still centered to be sure that its membrane was measured. If the cell moved, discard the data.Now go to the Auto Correlation window and manually delete individual repeats that contain zooming maneuvers, bleaching, large clusters, or other artifacts. Use a right click and select Delete to remove the artifacts. Selecting the actual artifacts requires practice.The removal of repeats is a simple process, but the repeats need to be assessed correctly to know what should be removed and which should not. Then save the final file in FCS format. Keep only cells that have at least four repeats left after the removal of artifacts for further analysis.A typical membrane receptor Auto correlation curve is smooth and has a transit time in the range of 10 to 400 milliseconds. Set the fitting domains so that both the start and the end of the steepest sloping portion, representing diffusion, is represented. Using a model of free diffusion in two dimensions to fit the data, pay attention to the most deeply-sloped portion of the curve.Small fluctuations at or around one second at the tau scale, seen in the residual, are acceptable. If the curve fits badly, but the cell has no apparent problems, such as moving, bleaching, or clusters, then the starting values or upper and lower limits of the model likely need changing. Once the model fits well to the Auto correlation curve, extract the values of the variable parameters.The number of molecules can vary between 0.5 to around 200 per square micron for endogenously-expressed proteins. Check that the CPM value from the cell measurement is at least 33%of that recorded from freely diffusing antibodies. After watching this video, you should have a good understanding of how to measure the diffusion rate of proteins using fluorescent correlation spectroscopy.Once mastered, this technique can be performed in five to six hours if performed properly. While attempting this procedure, it is important to keep the cells on ice until around 20 minutes before the start of measurement. Do not measure any samples more than two hours.Following this procedure, other methods like imaging can be performed in order to answer additional questions, such as how the diffusion rate correlates to the expression level of a certain protein. After its development, this technique paved the way for researchers in the fields of molecular biology and immunology to explore dynamics of proteins in live cells.

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10.8K Görüntüleme.  Karolinska Institutet. The overall goal of this protocol is to measure the diffusion rate of a labeled protein in membranes of primary immune cells using fluorescence correlation spectroscopy. This method can help identify key questions in the immunology and cell biology fields, such as identifying natural killer cells with a more active membrane receptor dynamics. The main advantage of this technique is that it is used with live cells, so it accurately reflects the natural state on how molecules move.The im...