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

Summary

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.

Abstract

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.

Introduction

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 quantifyi....

Protocol

1. Staining for FCS

1. Isolate murine NK cells from spleen lymphocytes using magnetic bead labelling, as per the manufacturer'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.
2. For murine cells expressing Fc receptors, block the Fc receptors with the antibody clone 2.4G2 at 5 µg/µl in 25 μl of phosphate-buffered saline....

Results

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 μm² 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 for f.......

Discussion

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.......

Materials

List of materials used in this article

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