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Single Cell Analysis of Lipid Rafts

  • William T. Lee
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 1066)

Abstract

Lipid rafts are plasma membrane microdomains that serve as platforms for the assembly of proteins involved in signal transduction pathways. Given that lipid rafts are relatively resistant to cold extraction with nonionic detergents, lipid raft associated and nonassociated proteins have been identified using biochemical methods such as sucrose-gradient density centrifugation. For identification of raft-associated proteins in individual cells, imaging methods, such as fluorescence microscopy, can be used. Detergent solubilization of non-raft regions of the plasma membrane and extraction of non-raft associated proteins are done on cells affixed to microscope slides and prior to immunostaining. This methodology has the advantages of requiring smaller cell numbers than traditional biochemical methods and also permits the study of migration of signaling proteins into and out of rafts during cell activation. An additional adaptation of the method allows identification of lipid raft-associated proteins during cognate interactions between cells. Here, as an example, we describe the methodology used in our laboratory to study lipid raft-associated molecules during T lymphocyte interactions with antigen-presenting cells.

Key words

Lipid rafts Plasma membrane Signal transduction Microscopy Cell–cell interactions T lymphocytes 

1 Introduction

Lipid rafts, also called glycolipid-enriched membranes or detergent-insoluble glycosphingolipid-enriched domains, have been studied in the context of neurotransmission, regulation of membrane fluidity, and also receptor signaling and receptor trafficking in different cell types (reviewed in [1]). However, most studies have focused on their role as platforms for assembling signaling clusters in activating immune cells (B and T lymphocytes). Lipid rafts are formed through enrichment of certain membrane lipids, particularly cholesterol, glycosphingolipids, and sphingomyelin, surrounded by unsaturated glycerophospholipids [2]. These specialized plasma membrane microdomains are more ordered and tightly packed than the surrounding bilayer, but float freely in the membrane. Although most membrane proteins are absent from lipid rafts, GPI-linked and acylated proteins are concentrated in these microdomains [2]. Thus, membrane proteins may either be always excluded or constitutively associated with lipid rafts. Alternatively, proteins may migrate into rafts during cell activation. While not without controversy [3, 4], it is generally believed that such regulation of the spatial association or, alternatively, prevention of association, of interacting proteins plays a critical role in productive signal transduction. For example, in T lymphocytes engagement of the receptor for antigen (TCR) and subsequent cell activation is spatially regulated at a highly organized region (immunological synapse) formed at the interface between the T cell and the antigen-presenting cell (APC) (reviewed in [5]). Lipid rafts also migrate to the immune synapse and several lines of evidence point to them being a key regulator of signaling: (1) Engagement of the TCR by antigen or anti-TCR antibodies leads to rapid raft aggregation [6]; (2) Several of the molecules necessary for T cell signaling and activation are constitutively associated or migrate to lipid rafts upon TCR engagement [1, 6]; (3) Signaling protein activation, as measured by tyrosine phosphorylation, occurs on lipid rafts [1, 3]; (4) Raft aggregation alone, by clustering GM1 gangliosides with cholera toxin B, results in TCR-associated signaling molecule recruitment and tyrosine phosphorylation [6]; (5) Inhibition of cell activation (clonal anergy) was associated with exclusion of essential signaling molecules from lipid rafts [7, 8].

Compared to non-raft elements of the plasma membrane, lipid rafts are resistant to extraction at low temperature by nonionic detergents, such as Triton X-100 (TX-100) [9, 10]. Thus, rafts and raft-associated proteins are typically identified after separation of membrane detergent lysates by density gradient centrifugation [10, 11]. However, large numbers of cells are required for this technique and an alternative approach is necessary when starting material, such as subpopulations of primary ex vivo cells, is limiting. When material is limiting, raft-associated proteins may be identified on single cells using microscopy. We have adapted a method initially described by Janes et al. to identify lipid raft-associated, TCR-signaling proteins in resting T cells [6]. The cells of interest are affixed to microscope slides and non-raft proteins are extracted before staining the cells for target proteins of interest. Thus, by comparing cells that have been treated with detergent to cells that have not been treated, raft-associated proteins (present after treatment) and raft-excluded proteins (absent after treatment) can be identified. We found that cells of different differentiation stages (e.g., naive versus memory T cells) have different patterns of constitutive raft-associated signaling assemblies [12]. For example, Fig. 1 shows two signaling proteins (TCR and CD45) that are constitutive raft-associated proteins in memory cells (as indicated by resistance to detergent extraction) but not naive cells. This pattern might reflect the different activation capabilities of the two cell types. We have further extended this method to examine individual T cells during their cognate interactions with APCs and subsequent activation by specific antigen [8, 12]. We showed that regulation of lipid raft assemblies, namely exclusion of critical signaling molecules, consequently controls productive signal transduction and cell activation [8]. For example, during activation of memory T cells the critical signaling protein ZAP-70 associates with lipid rafts [1] and migrates to the immune synapse ([13] and Fig. 2). However, in memory cells exposed to a toxin that causes them to be inactivated (anergic), ZAP-70 is excluded from both lipid rafts and the immunological synapse ([8] and Fig. 2). Thus, cell activation is blocked by the physical separation of key signaling proteins.
Fig. 1

Cell-type differences in constitutive raft-associated proteins. Mouse naive and memory T cells were labeled with cholera toxin-B-subunit (CTB)-rhodamine to identify lipid rafts (GM1). The cells were either (control) stained with fluorescent-labeled antibodies (as described in Subheading 3) or (1 % TX-100) permeabilized with 1 % Triton X-100 (to remove non-raft proteins) before staining. The antibodies were directed against two membrane proteins involved in cell signaling (TCR or CD45). Staining is shown with conversion to grayscale. The far-right column shows the differential interference contrast (DIC) images of cells

Fig. 2

Signaling blockade is associated with exclusion from lipid rafts. Memory CD4+ T cells were labeled with CTB-rhodamine (GM1) and were conjugated with antigen-presenting cells pre-pulsed with either (Stimulated ) specific antigen, or (Anergic ) an inactivating toxin. The cells were either (control ) stained with fluorescent-labeled antibodies, or (1 % TX-100 ) permeabilized with 1 % Triton X-100 before staining. The antibodies were directed against two essential signaling proteins (TCR or ZAP-70). Staining is shown with conversion to grayscale. The far-right column shows the DIC images of the conjugates

Here we describe our laboratory’s procedure for identification of lipid rafts and raft associated proteins in individual resting T cells or interacting couplets of T cells and APCs. This method relies upon the specific identification of the signaling protein of interest by staining with fluoresceinated antibodies before or after membrane extraction with cold, nonionic detergent, followed by fluorescence microscopy. Although our focus is on immune cells, the tech-niques are adaptable to other cell types and cell–cell interactions. Furthermore, the microscopy technique also allows for examination of dynamic events occurring in individual cells (or during “cross-talk” between interacting cells) as opposed to bulk populations.

2 Materials

All solutions and equipment coming into contact with cells, at least through the cell culture stage, must be sterile. Prepare all solutions using Milli-Q-purified water or equivalent. Prepare and store all reagents at 4 °C, unless otherwise indicated.

2.1 Cell Culture

  1. 1.

    Cells: primary ex vivo cells or tissue culture cells (see Note 1).

     
  2. 2.

    Tissue culture medium: complete RPMI-10 medium (see Note 2). RPMI-10 medium is enriched by the addition (to final concentrations) of Fetal Bovine Serum (FBS) (10 %), l-glutamine (2 mM), 2-mercaptoethanol (50 μM), penicillin (100 U/ml), and streptomycin sulfate (100 μg/ml).

     
  3. 3.

    Stimulus to be tested.

     
  4. 4.

    12-well tissue culture plates.

     

2.2 Slide Preparation

  1. 1.

    12-well multitest slides.

     
  2. 2.

    70 % ethanol.

     
  3. 3.

    0.01 % poly-l-Lysine solution: 0.1 % poly-l-Lysine solution diluted 1:10 with deionized water (see Note 3).

     
  4. 4.

    Phosphate-Buffered Saline (PBS).

     

2.3 Lipid Raft Identification and Cell Staining

  1. 1.

    PBS/1 % bovine serum albumin (BSA) and PBS/0.1 % BSA.

     
  2. 2.

    Triton X-100 extraction buffers (2 detergent percentages): 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EGTA, 5 mM EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 mM iodoacetamide, 5 mM benzamidine HCl, 1 mM AEBSF, 50 mM NaF (optional, see Note 4), 10 mM Na4P2O7 (optional), 1 mM Na3VO4 (optional), add Triton X-100 to either 1 % (v/v) or 0.2 % (v/v). The Triton X-100 solutions, without the protease inhibitors, can be stored for a year or longer at 4 °C. Add the protease inhibitors immediately before use from 100× stocks in water. Store the protease inhibitor stocks for up to 3–4 months at −20 °C.

     
  3. 3.

    0.4 % paraformaldehyde solution: 4 g paraformaldehyde (electron microscopy grade) in 100 ml of PBS. Dissolve paraformaldehyde by heating solution to 70 °C in a fume hood for 1 h. Cool to room temperature and adjust pH to 7.2 with 0.1 M NaOH or 0.1 M HCl. Store protected from light for up to 2 weeks at 4 °C.

     
  4. 4.

    Pre-titrated (optimal) primary or directly conjugated fluorescent antibody (see Note 5).

     
  5. 5.

    Pre-titrated (optimal) fluorescent secondary antibody (optional).

     
  6. 6.

    Rhodamine (TRITC)-conjugated cholera toxin B subunit (CTB) (List Biological Laboratories, Campbell, CA, USA) or Alexa Fluor 594-conjugated CTB (Invitrogen, Grand Island, NY, USA) diluted to 10 μg/ml in PBS/0.1 % BSA (optional, see Note 6).

     
  7. 7.

    Anti-CTB antibody (EMD Millipore, Billerica, MA, USA), used at dilution suggested by manufacturer (optional, see Note 6).

     
  8. 8.

    10 mM Methyl-β-cyclodextrin (MβCD) (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS (optional, see Note 7).

     
  9. 9.

    SlowFade Light Antifade Kit (Invitrogen).

     

3 Methods

Similar to the density gradient centrifugation technique, rafts and raft-associated proteins are identified by resistance to Triton X-100 solubilization. Cells are treated with detergent after adherence to microscope slides and solubilized non-raft proteins are removed by washing. Hence, fluorescent microscopy examination of individual proteins before and after exposure to Triton X-100 indicates which proteins are associated with lipid rafts. An immunological example (T lymphocytes) is described; however, the basic procedure may be adapted to other cell types.

3.1 Slide Preparation

  1. 1.

    Set up four wash chambers that are large enough to hold the desired number of slides to be coated with poly-l-lysine (see Note 3). Slides should be laid flat in the chambers and can be manipulated by using forceps to grab onto one corner of the slide. The wash chambers should be filled with enough volume to fully cover the slides and the individual chambers should contain: (1) 70 % ethanol; (2) deionized water; (3) 0.01 % poly-l-lysine solution; (4) PBS.

     
  2. 2.

    Submerge the 12-well multitest slides into the chamber containing 70 % ethanol and incubate for 5 min at room temperature.

     
  3. 3.

    Remove the slides and transfer into the chamber containing deionized water and incubate for 5 min at room temperature.

     
  4. 4.

    Transfer the slides into the poly-l-lysine solution and incubate for 5 min at room temperature.

     
  5. 5.

    Remove the slides and air dry.

     
  6. 6.

    Repeat the poly-l-lysine incubation and air-drying step for a total of three incubation periods.

     
  7. 7.

    Transfer the slides to the chamber containing PBS and incubate for 5 min at room temperature.

     

3.2 Lipid Raft Identification in Single Cells

This procedure for examination of individual cells is useful for determining if molecules of interest are constitutively associated with lipid rafts in nonactivated cells. The procedure may also be applied to cells after cell activation or receptor–ligand interaction to determine whether the molecule of interest migrates to rafts during signaling.
  1. 1.

    Prepare the primary (or immune) cells of interest for study (see Note 1). Alternatively, collect cells from tissue culture (see Note 8). In these procedures we will use T cells as our model cell type.

     
  2. 2.

    (Optional) If lipid rafts are first labeled with fluorescent CTB for identification of the GM1 ganglioside component of rafts, then resuspend the T cells to 2 × 107/ml in PBS/0.1 % BSA in a 15 ml centrifuge tube (see Notes 6 and 9). Then add CTB-rhodamine or CTB-Alexa Fluor 594 to 10 μg/ml. Incubate the tube for 30 min on ice. Fill the centrifuge tube PBS/0.1 % BSA and centrifuge at 200 × g for 10 min. Repeat the wash step. Wash an additional three times with 15 ml of PBS (no BSA). If CTB labeling is not desired, then omit this step and move to step 3.

     
  3. 3.

    Prepare the slides by washing the cells, if they were not washed in step 2, with 15 ml of PBS (no BSA). Resuspend the T cells to 2 × 107/ml in PBS and then pipet 20 μl of cells into each well of the poly-l-lysine-coated 12-well multitest slides and then incubate for 20 min in a 37 °C humidified incubator (see Note 10). Use forceps to pick the slides up at one corner and, using a squirt bottle containing a washing buffer (e.g., PBS), project a steady stream onto the slide surface for a minimum of 10 s. Shake the slide free of wash buffer. This constitutes a single wash, which should be repeated for a total of three washes.

     
  4. 4.

    (Optional) If patching or aggregation of lipid rafts is desired (see Note 6), then add this step into your protocol. If not desired, then proceed to step 5. To patch lipid rafts, wash the slides once with PBS/0.1 % BSA, as described in step 3, and then add 15 ml of anti-CTB antibody (1:250 dilution of the commercial preparation in PBS/0.1 % BSA) to the sample well(s). Incubate the slides for 30 min on ice. Raise the temperature of the cells by moving the slides to 37 °C and continue incubating for an additional 20 min.

     
  5. 5.

    Extract the non-raft proteins by selecting wells designated as the control samples and the wells designated as the test samples (see Note 11). For control samples, fix the cells by adding 15 μl of 4 % paraformaldehyde to each well and then incubating the slides for 20 min at room temperature. Wash the slides once with PBS as described in step 3, and then permeabilize the cells by adding 15 μl/well of the 0.2 % TX-100 solution. Incubate the slides for 5 min at 4 °C and then wash the slides once with PBS as described in step 3. (Optional) If MβCD treatment is to be done (see Note 7), prior to the cell fixation step, incubate the slides with a 10 mM MβCD solution for 30 min at 37 °C and wash the slides twice with PBS as described in step 3.

     
  6. 6.

    For test samples, permeabilize the cells by adding 15 μl/well of the 1 % TX-100 solution and incubating the slide for 5 min at 4 °C. Wash the slides once with PBS as described in step 3. Fix the cells by adding 15 μl of 4 % paraformaldehyde to each well and then incubating the slides for 20 min at room temperature. Wash the slides once with PBS as described in step 3. (Optional) If MβCD treatment is to be done (see Note 7), prior to permeabilization with the 1 % TX-100 solution, incubate the slides with a 10 mM MβCD solution for 30 min at 37 °C and wash the slides twice with PBS as described in step 3.

     
  7. 7.

    To identify proteins by immunostaining, first block the slides by incubating them PBS/1 % BSA overnight at 4 °C (see Note 12). The next day, bring the slides to room temperature and wash them twice with PBS as described in step 3. Stain the cells with 15 μl of either the unlabeled primary antibody or the directly conjugated fluorescent antibody at the optimal dilution for 1 h at room temperature (see Note 13). Wash slides three times with PBS as described in step 3. If you are using indirect staining, then stain the cells with 15 μl of the fluorescent secondary antibody at its optimal dilution for 1 h at room temperature. Wash the slides three times with PBS as described in step 3 and then mount the coverslips using the SlowFade Antifade Kit according to the manufacturer’s instructions. Store the slides in the dark at 4 °C until microscopy analysis (see Note 14).

     
  8. 8.

    Analyze cells using fluorescent-based microscopy (see Note 15). Compare extraction control and test samples for each cell treatment group.

     

3.3 Lipid Raft Identification During Cell–Cell Interactions

T cell stimulation by peptide antigen requires that the T cell physically interact with another cell (APC) for proper activation. This procedure allows for the study of movement of lipid rafts and the migration of raft-associated proteins during cell–cell interactions. The steps followed in this method are similar to those used in Subheading 3.2 above, except that prior to processing for microscopy, cell–cell conjugates are formed. APCs bind to specific antigen and then couple with specific T cells to induce T cell activation. Conjugates containing both cell types are formed and then adhered to microscope slides and cultured to permit cell signaling.
  1. 1.

    Prepare cells of interest for study (see Note 1). In this immunological example, T cells must recognize the specific antigen that will be presented and APCs must be MHC histocompatible (see Note 16). T cells and APCs are prepared separately. Primary CD4+ T cells or T cell subsets are prepared. Alternatively, collect and process T cells from tissue culture. Primary APCs are prepared or collected from tissue culture. Either cell type may be held in complete RPMI-10 medium on ice in 15 ml conical tubes until used.

     
  2. 2.

    (Optional) If lipid rafts are first labeled with fluorescent CTB for identification of the GM1 ganglioside component of rafts, then only label the cell type of interest (e.g. T cells), while leaving the partner cell population unlabeled. Thus, resuspend the T cells to 2 × 107/ml in PBS/0.1 % BSA in a 15 ml centrifuge tube (see Notes 6 and 9). Then add CTB-rhodamine or CTB-Alexa Fluor 594 to a concentration of 10 μg/ml. Incubate the tube for 30 min on ice. Fill the centrifuge tube with PBS/0.1 % BSA and centrifuge at 200 × g for 10 min. Repeat the wash step and then wash three more times with 15 ml of PBS (no BSA). If CTB labeling is not desired, omit this step and move to step 3.

     
  3. 3.

    To pulse the APCs with antigen, resuspend the cells to 1 × 107/ml in prewarmed 37 °C complete RPMI-10 medium with 1 μg/ml of peptide antigen and add the cells into individual wells of a 24-well tissue culture plate (1 ml/well) (see Note 17). Incubate the culture plate for 2 h at 37 °C in a 5 % CO2 tissue culture incubator (see Note 10). After the incubation period, collect the cells and transfer them into a 15 ml tube. Centrifuge for 10 min at 200 × g at 4 °C. Resuspend the cells in 15 ml PBS and centrifuge again for 10 min at 200 × g, 4 °C. Resuspend the APCs to 4 × 107/ml in PBS.

     
  4. 4.

    Resuspend the T cells to 2 × 107/ml in prewarmed (37 °C) PBS.

     
  5. 5.

    To form conjugates between the antigen-specific T cells and the antigen-pulsed (or control) APCs, add 12.5 μl antigen-pulsed APCs and 12.5 μl of T cells to 1.5 ml microcentrifuge tubes and gently pipet up and down several times to mix the cells. Centrifuge the tubes at 400 × g for 10 s at room temperature. Quickly and carefully pipet 10 μl of cells onto prewarmed (37 °C) poly-l-lysine coated wells of a 12-well multitest slide and incubate at 37 °C for various lengths of time (0–60 min) (see Note 18). Wash the slides once with PBS as described in Subheading 3.2, step 3.

     
  6. 6.

    Identify samples not only as stimulation test samples and controls but also as paired samples for non-raft protein extraction test samples and control samples as described in Subheading 3.2, step 5 (see Note 11). For extraction control samples, fix the cell conjugates by adding 15 μl of 4 % paraformaldehyde to each well and then incubating the slides for 20 min at room temperature. Wash the slides once with PBS as described in Subheading 3.2, step 3, then permeabilize the cells by adding 15 μl/well of the 0.2 % TX-100 solution. Incubate the slides for 5 min at 4 °C and then wash the slides once with PBS as described in Subheading 3.2, step 3 (see Note 19).

     
  7. 7.

    For test samples, first permeabilize the cell conjugates by adding 15 μl/well of the 1 % TX-100 solution and incubating the slide for 5 min at 4 °C. Wash the slides once with PBS as described in Subheading 3.2, step 3, then fix the cells by adding 15 μl of 4 % paraformaldehyde to each well and incubating the slides for 20 min at room temperature. Wash the slides once with PBS as described in Subheading 3.2, step 3.

     
  8. 8.

    To identify proteins in the conjugates by immunostaining, first block the slides by incubating them PBS/1 % BSA overnight at 4 °C (see Note 12). The next day, bring the slides to room temperature and wash them twice with PBS as described in Subheading 3.2, step 3. Stain the cells with 15 μl of either the unlabeled primary antibody or the directly conjugated fluorescent antibody at the optimal dilution for 1 h at room temperature (see Note 13). Wash the slides three times with PBS as described in Subheading 3.2, step 3. If using indirect staining, then stain the cells with 15 μl of the fluorescent secondary (detecting) antibody at its optimal dilution for 1 h at room temperature. Wash the slides three times with PBS as described in Subheading 3.2, step 3 and then mount the coverslips using the SlowFade Antifade Kit according to the manufacturer’s instructions. Store the slides in the dark at 4 °C until microscopy analysis (see Note 14).

     
  9. 9.

    Analyze by fluorescence-based microscopy (see Note 15). Compare extraction control and test samples for each cell treatment group.

     

4 Notes

  1. 1.

    Obviously this will vary depending on the individual laboratory’s specific experimental model. In our laboratory, we generally begin with single cell suspensions from murine spleen or lymph nodes after removal of red blood cells. Most often, because our own studies focus on antigen-driven T lymphocyte responses, we enrich our starting populations for either CD45RBlo (antigen-experienced or memory) and CD45RBhi (naive) CD4 T cells, using methods that we previously have described [12, 14]. Very few cells are needed for an individual test.

     
  2. 2.

    The percentage of serum used in Subheading 3 is indicated by medium-numeral. In this example, RPMI-10 indicates that FBS is added to 10 %. If no numeral is added to the base name, no serum is added. The FBS is heat inactivated for 1 h at 56 °C and stored at 4 °C before use. The base medium, RPMI, is routinely used in our laboratory for tissue culture; we have not tested other media types but it is likely that the routine culture media for the user is suitable for this procedure.

     
  3. 3.

    Coating the 12-well multitest slides with poly-l-lysine will increase cell attachment to the slide. The volume of poly-l-lysine solution will vary depending upon the number of slides to be coated. The slides will be immersed in the solution, so sufficient coating solution should be prepared to cover all slides to be used in the experiment.

     
  4. 4.

    Phosphatase inhibitors (NaF, Na4P2O7, Na3VO4) should be included when examining phosphoproteins or raft associations that occur as a consequence of kinase activity.

     
  5. 5.

    We routinely use Alexa dyes because of their brighter and more stable fluorescence as compared to other dyes. However, we have had good success using more commonly used dyes such as FITC, Rhodamine, and Cy5. Specific dilutions of the antibody reagent will vary depending upon the antigen target, manufacturer, etc., so precise concentrations are not listed here. However, all of the antibody reagents, and especially, due to their high fluorescence intensity, the Alexa-conjugated reagents, should be pre-titrated prior to assay for use at optimal concentrations.

     
  6. 6.

    Cholera toxin B subunit binds to GM1 gangliosides, which are integral components of lipid rafts [15], and therefore, using fluorescent CTB will permit visual identification by fluorescent microscopy. Generally the labeling will show a homogeneous distribution pattern because of the small size of lipid rafts (<70 nm in diameter [16]). Aggregating rafts with anti-CTB causes a patched distribution and often a better visualization of colocalization of raft-associated proteins can be seen [6]. Of note, Janes et al. demonstrated that (warm) raft aggregation by anti-CTB antibodies can promote receptor signaling [1]. We routinely include fluoresceinated CTB either along with antibodies to our protein of interest (e.g. Fig. 1) or in separate samples as part of our control group.

     
  7. 7.
    Lipid raft-associated proteins, unlike cytoskeletal or detergent-insoluble proteins, become detergent soluble in the absence of lipid rafts [17]. MβCD disrupts lipid rafts by depleting membrane cholesterol and this permits solubilization of raft-associated proteins by TX-100. We often include this treatment of cells as an additional control when determining if a specific protein is a raft protein as opposed to a protein attached to the cytoskeleton because in the former case, the protein will no longer be visualized after TX-100 treatment if the cells are first exposed to MβCD. Figure 3 shows staining of GM1 gangliosides with CTB-rhodamine with and without MβCD prior to TX-100 extraction.
    Fig. 3

    MβCD disrupts lipid rafts. T cells were labeled with CTB-rhodamine to identify lipid rafts (GM1). The cells were left untreated or treated with 10 mM MβCD for 30 min at 37 °C before analysis. Staining is shown with conversion to grayscale. The second and fourth columns show the DIC images of the cells

     
  8. 8.

    Cell lines carried in vitro are not generally resting cells. Thus, a given experiment may be influenced by variances in culture conditions and cell growth. For T cell lines, we generally collect cells >7 days following the last stimulation period with antigen so that the cells might more reasonably reflect a resting or unstimulated cell.

     
  9. 9.

    The volumes and size containers described in this procedure will vary depending on the individual experiment. For our studies, we generally obtain 2.5–3.5 × 107 CD4+ T cells per one mouse spleen and may only obtain 5 × 106 CD4+ memory cells from several spleens. For our purposes, the volumes and container sizes noted in Subheading 3 are generally sufficient for our needs, including the higher volumes used for washing steps. However, these can both be scaled up depending upon the requirements of the specific experiment.

     
  10. 10.

    For all 37 °C incubations, we use our standard 5 % CO2 culture incubator. The CO2 is not a necessary component to the staining incubations; we use the incubator out of convenience. For any ligand stimulation or activation of the cells prior to preparation of the cells for lipid raft analysis, the tissue culture incubator is an essential component.

     
  11. 11.

    Lipid raft-associated proteins will be indicated by resistance to extraction with 1 % TX-100. In our studies, wells containing identical samples are divided into two groups to compare staining of cells with and without extraction. In Subheading 3, “control samples” refer to the staining and identification of the protein of interest in samples without extraction, whereas, “test samples” refer to the staining and identification of the same protein after extraction. For control samples, a low concentration (0.2 %) of TX-100 is added prior to the fixation step to allow access of the staining antibodies to intracellular proteins. This concentration of detergent does not cause extraction of non-raft proteins. It is essential that all reagents be kept at cold temperatures (<4 °C) and membrane solubilization must be done on ice. At higher temperatures, raft proteins become increasingly soluble [18].

     
  12. 12.

    Although we usually perform an overnight blocking step in order to minimize nonspecific binding before immunostaining, it may be possible to reduce this blocking period by several hours by performing this step at room temperature.

     
  13. 13.
    Control samples should be carefully chosen and included. Important controls would be the inclusion of lipid raft measurements (e.g., CTB) and also known raft-associated proteins and known non-raft proteins. For T cell analyses, we and others have found that the proteins LAT and CD71 (transferrin receptor) work well as indicators of raft and non-raft proteins, respectively [6, 10, 12]. Figure 4 shows an example of immunostaining for LAT and CD71 with and without extraction with 1 % TX-100.
    Fig. 4

    Lipid raft controls. T cells were labeled with CTB-rhodamine to identify lipid rafts (GM1). The cells were either (control) stained with fluorescent-labeled antibodies or (1 % TX-100) permeabilized with 1 % Triton X-100 (to remove non-raft proteins) before staining. The antibodies were directed against a constitutively raft-associated protein (LAT) or a constitutively raft-excluded protein (CD71). Staining is shown with conversion to grayscale. The far-right column shows the differential interference contrast (DIC) images of cells

     
  14. 14.

    In our experience, fluorescence stability is enhanced for any of the labels by the use of a commercial Slow-Fade reagent. We have also found that the fluorescence integrity of the prepared slides is relatively stable when they are stored in the dark at 4 °C. However, for raft analyses the prior detergent extraction may lead to some sample instability and it is recommended that the samples be analyzed within a few days after preparation.

     
  15. 15.

    Analysis requires access to confocal or epifluorescence microscopes and deconvolution software. In our experiments, we generally acquire images using a Hamamatsu ORCA-ER digital CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan) attached to a Zeiss Axioskop2 mot plus microscope (Carl Zeiss, Gottingen, Germany), using OpenLab software (Improvision Inc, Lexington, MA, USA).

     
  16. 16.

    For primary cells, wild-type, conventional mice possess too low a frequency of antigen-specific cells to be useful for this procedure. For this reason, T cells which are derived from mice transgenic for a specific TCR are most amenable for study. However, mice transgenic for a given TCRβ chain or even nontransgenic mice may be used if the presenting antigen is a bacterial superantigen [19]. Owing to the high frequency of superantigen-reactive T cells [19], cell conjugates should be visualized. Cloned T cells or antigen-specific T cell lines can also be easily used in this assay. For the analysis of CD4+ T cell-APC conjugates, APCs are prepared by treatment of murine red blood cell-depleted spleen cells with anti-Thy-1 plus baby rabbit complement to deplete T cells and enrich for B cells and macrophages [14].

     
  17. 17.

    The exact dose of antigen or other stimulus will vary for the specific antigen or stimulus. The concentration used in this procedure is for stimulation with a specific peptide antigen and was chosen so that it would lead to a strong binding interaction between the T cell and APC. A whole antigen which requires processing into peptides might require ten or even a hundred-fold higher amount to facilitate cell coupling. In our studies, we include control APCs that are either pulsed with an irrelevant (to the specific T cell of interest) peptide or unpulsed.

     
  18. 18.

    Because the T cell–APC interactions and cell signaling are dynamic events, kinetic experiments are generally done where the length of time that the T cell and APC are cultured together is varied (from seconds to hours). During this period, many molecules on both the T cell and the corresponding APC will migrate into defined membrane regions (immunological synapse) to interact in a highly organized fashion with other molecules on the same cell or on the opposing cell. Lipid rafts, visualized by CTB binding to GM1 gangliosides, also migrate into the immunological synapse [12, 20]. In this way, signaling complexes are built and signal transduction occurs in both the T cell and APC.

     
  19. 19.

    If desired, the cells can be treated with MβCD prior to extraction so that lipid rafts are disrupted and raft-association of the protein of interest is confirmed. The procedure steps would be done as described in Subheading 3.2, steps 5 and 6 (also see Note 7).

     

Notes

Acknowledgment

This work was supported by National Institutes of Health (AI35583).

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Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • William T. Lee
    • 1
  1. 1.The Laboratory of ImmunologyNew York Department of Health, The Wadsworth CenterAlbanyUSA

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