Studying Organelle Dynamics in B Cells During Immune Synapse Formation

     NOTE: The following steps were performed using IIA1.6 B cells.

1. B cell activation with antigen-coated beads

1. Preparation of antigen-coated beads
   1. To activate B cells, use NH₂-beads covalently coated with antigen (Ag-coated beads), which are prepared using 50 µL (~20 x 10⁶ beads) of 3 µm NH₂-beads with activating (BCR-ligand+) or non-activating (BCR-ligand-) antigens.
   2. For IIA1.6 B cells use anti-IgG-F(ab')₂ fragment as BCR-ligand+ and anti-IgM-F(ab')₂ or bovine serum albumin (BSA) as BCR-ligand-. To activate primary B cells or IgM⁺ B cell line use anti-IgM-F(ab')₂ as BCR-ligand+ and BSA as BCR-ligand-.
      NOTE: To avoid the binding of ligands to the Fc receptor, use F(ab') or F(ab')₂ antibodies fragments instead of full-length antibodies.
   3. To proceed with the preparation of Ag-coated beads, place the beads in low protein binding microcentrifuge tubes to maximize the beads recovery during the sample manipulation. Add 1 mL of 1x phosphate-buffered saline (PBS) to wash beads and centrifuge at 16,000 x g for 5 min. Aspirate the supernatant.
   4. Resuspend the beads with 500 µL of 8% glutaraldehyde to activate the NH₂ groups and rotate for 4 h at room temperature (RT).
      CAUTION: The glutaraldehyde stock solution should only be used in a chemical fume hood. Follow the instructions on the material safety data sheet (MSDS).
   5. Centrifuge beads at 16,000 x g for 5 min, remove the glutaraldehyde and wash the beads three more times with 1 mL of 1x PBS.
      CAUTION: The glutaraldehyde solution should be discarded as a hazardous chemical waste.
   6. Resuspend the activated beads in 100 µL of 1x PBS. The sample can be divided into two low protein binding microcentrifuge tubes: 50 µL for the BCR-ligand+ and 50 µL for BCR-ligand-.
   7. To prepare the antigen solution use two 2 mL low protein binding microcentrifuge tubes containing 100 µg/mL of antigen solution in 150 µL of PBS: One tube with BCR-ligand+ and other with BCR-ligand-.
   8. Add 50 µL of activated beads solution to each tube containing 150 µL of antigen solution, vortex and rotate overnight at 4 °C.
   9. Add 500 µL of 10 mg/mL BSA to block the remaining reactive NH₂ groups on the beads and rotate for 1 h at 4 °C.
   10. Centrifuge the beads at 16,000 x g for 5 min at 4 °C and remove the supernatant. Wash the beads with cold 1x PBS three more times.
   11. Resuspend the activated beads in 70 µL of 1x PBS.
   12. To determine the final concentration of Ag-coated beads, dilute a small volume of beads in PBS (1:200) and count using a hemocytometer. Then store at 4 °C until use.
       NOTE: Ag-coated beads should not be stored for more than 1 month.
2. Preparation of poly-L-lysine coverslips
   1. Before the B cell activation assay with Ag-coated beads, prepare poly-L-lysine-coated coverslips (PLL-coverslips). Use a 50 mL tube containing 40 mL of 0.01% w/v of PLL solution and immerse the 12 mm-diameter coverslips into the solution. Rotate overnight at RT.
   2. Wash the coverslips with 1x PBS and leave to dry on a 24-well plate lid covered with paraffin film. Proceed with B cell activation.
3. B cell activation
   1. To start B cell activation with beads, first dilute the IIA1.6 B cell line to 1.5 x 10⁶ cells/mL in CLICK medium (RPMI-1640 supplemented with 2 mM L-Alanine-L-Glutamine + 55 µM beta-mercaptoethanol + 1 mM pyruvate + 100 U/mL Penicillin + 100 µg/mL streptomycin) + 5% heat-inactivated fetal bovine serum [FBS]).
   2. Combine 100 µL (150,000 cells) of IIA1.6 B cells with Ag-coated beads at 1:1 ratio in 0.6 mL tubes. Mix gently using a vortex and seed onto PLL-coverslips. Incubate for different time points in a cell culture incubator (37 °C / 5% CO₂). The typical activating time points are 0, 30, 60 and 120 min. For time 0, place the PLL-coverslips into the 24-well plate lid on ice. Add the cells-Ag-coated bead mixture and incubate for 5 min on ice.
      NOTE: It is important to mix by vortex instead of pipetting up and down, because this could reduce the number of beads in the sample, due to their accumulation in the plastic tip of the pipette. Use beads coated with BCR-ligand- as a negative control for each assay. We recommend activating the longest incubation time first and then the following time points. Calculate intervals of activation for samples such that they are ready for fixation at the same time.
   3. Add 100 µL of cold 1x PBS to each coverslip to stop the activation and continue with the immunofluorescence protocol. See protocol section 3.

2. B cell activation on antigen-coated coverslips

1. Preparation of antigen-coated coverslips
   1. Before activation, prepare the antigen solution (1x PBS containing 10 µg/mL BCR-ligand+ and 0.5 µg/mL rat anti-mouse CD45R/B220).
      NOTE: Consider preparing the coverslips the day before the assay. B220 improves B cell adhesion, however, the BCR-Ligand+ is sufficient to generate the spreading response.
   2. Place the 12 mm coverslip onto a 24-well plate lid covered with paraffin film, add 40 µL of antigen solution onto each coverslip and incubate at 4 °C overnight. Seal the plate to avoid evaporation of antigen solution.
   3. Wash the coverslips with 1x PBS and air dry.
2. B cell activation
   1. To start the activation, dilute IIA1.6 B cells to 1.5 x 10⁶ cells/mL in CLICK medium + 5% FBS.
   2. Add 100 µL of cells onto an antigen-coated coverslip (Ag-coverslip) and activate for different time points in a cell incubator at 37 °C / 5% CO2. The typical activating time points are 0, 30 and 60 min.
      NOTE: As in section 1, we recommend starting with the longest activating time point in order to fix all the samples at the same time.
   3. For time 0, place the 24-well plate lid containing the Ag-coverslip on ice and add the cells. Incubate for 5 min on ice.
   4. Carefully aspirate the media on each Ag-coverslip and then add 100 µL of cold 1x PBS to stop the activation. Continue with the immunofluorescence protocol. See section 3.

3. Immunofluorescence

NOTE: To avoid cross-reactivity of the secondary antibody with the BCR of IIA 1.6 B cells, do not use mouse-derived primary antibodies.

1. Remove the 1x PBS and proceed with the fixation of each coverslip.
   NOTE: To decide which fixation medium to use, check the antibody/dye data sheet. For example, for actin labeling by phalloidin use paraformaldehyde (PFA) fixation medium, and for the centrosome labeling by pericentrin use methanol fixation.
   1. Add 50 µL of 1x PBS supplemented with 4% PFA and incubate for 10 min at RT.
      CAUTION: Formaldehyde is toxic. Please read the MSDS before working with this chemical. PFA solutions should be only made under a chemical fume hood wearing gloves and safety glasses. PFA solution should be discarded as a hazardous chemical waste.
   2. Alternatively, add 50 µL of cold methanol and incubate for 20 min.
2. Wash three times with 1x PBS.
   NOTE: Coverslips can be stored at 4 °C at this point for maximum three days in 1x PBS.
3. Remove 1x PBS and add 50 µL of blocking buffer (2% BSA + 0.3 M glycine in 1x PBS) onto each coverslip. Incubate at RT for 10 min.
4. Aspirate gently and add 50 µL of permeabilization buffer (PB) (0.2% BSA + 0.05% saponin in 1x PBS). Incubate at RT for 20 min.
5. Prepare the antibodies or dyes in PB (use 30 µL per coverslip) and incubate at RT for 1 h or overnight at 4 °C. Refer to the Table of Materials for additional details.
   1. To label the microtubule organizing center (MTOC) or centrosome use the following antibodies: anti-γ-Tubulin (1:500), anti-Cep55 (1:500), anti-α-tubulin (1:500), anti-pericentrin (1:1000).
      NOTE: For centrosome labeling B cells can be transfected with a centrin-GFP expression plasmid.
   2. For labeling Golgi apparatus use anti-Rab6a (1:500).
      NOTE: Other antibodies or dyes can also be used.
   3. For labeling lysosomes, use anti-Lamp1 (1:200).
      NOTE: Anti-H2-DM and anti-MHC-II also can be used to label antigen processing compartments^15,16.
   4. For labeling endoplasmic reticulum use anti-Sec61a (1:500).
   5. For actin cytoskeleton consider the following: polymerized actin can be visualized by Phalloidin conjugated to fluorescent dyes.
      NOTE: B cells transfected with a LifeAct-GFP/RFP expression plasmid can also be used to label actin.
6. Wash the coverslips three times with PBS.
7. Dilute the secondary antibody or dyes in PBS (use 30 µL per coverslip) and incubate 1 h at RT.
   NOTE: Avoid exposing the samples to direct light to preserve the quality of the fluorescence signal.
8. Wash the coverslips three times with PBS.
9. Remove the PBS solution from coverslips.
10. Add 4 µL of mounting reagent to a microscope slide. Mount the coverslip onto the slide with the cell side facing down. Allow the slides to dry for 30 min at 37 °C or at RT overnight.
    NOTE: Consider using an "anti-fade" mounting reagent (see the Table of Materials).
11. Acquire fluorescence images on a confocal or epifluorescence microscope with a 60x or 100x oil immersion objective. For each acquisition consider the transmitted light or bright field to easily identify B cells interacting with beads.
    NOTE: Take three-dimensional (3D) images, covering the entire cell using z-stacks. We recommend taking 0.5 µm thick stacks, however this depends on the microscope.

4. Image analysis

NOTE: The following algorithms are described for ImageJ software. However, this can be performed using an equivalent software. Also, consider that for all fluorescence intensity measurements we use the integrated fluorescence density ("RawIntDen" in ImageJ), because this parameter considers the total amount of fluorescence in each pixel of the image, taking the area into account.

1. Analysis of the distribution of organelles in B cells activated with Ag-coated beads
   NOTE: To quantify the polarization of cell components to the IS, we define an arbitrary value as a measure of proximity to the IS. The index ranges between -1 (anti-polarized) and 1 (Fully polarized, object on the bead), as was previously presented by Reversat et al.¹².
   1. Estimate the polarity index for the centrosome and Golgi apparatus (Figure 1B).
      NOTE: This algorithm can be used for organelles that are confined to a one point.
      1. First define the bead and cell areas to analyze using the circle tool selection to delimit the boundaries of both, and then save them as regions of interest (ROI). See Figure 1, Figure 2, Figure 3, and Figure 4.
      2. Determine the cell center (CC) and the bead center (BC) by running Analyze | Measure on cell and bead areas respectively. X and Y values obtained from the Resultados window determine the center coordinates.
      3. Manually determine the center of the centrosome or Golgi apparatus (Organelle) using the point tool selection in ImageJ and run Analyze | Measure. X and Y values obtained from the Resultados window determine the coordinates.
      4. Then, draw an angle from CC to Organelle (a) and CC to BC (b) using the angle tool selection and run Analyze | Measure. The angle value in the results window shows the angle (α) between both vectors (a and b).
      5. Calculate the polarity index using the following formula:
         
   2. Estimate the polarity index for lysosomes (Figure 1F).
      NOTE: We use this algorithm to analyze the polarity of organelles that display a more dispersed distribution, such as lysosomes.
      1. Define the bead and cell areas to analyze, using the circle tool selection to delimit the boundaries of both, and save them as ROI. Once the bead and cell areas have been determind, set the fluorescence channel and project the image into one z-stack (Image | Stacks | Z-Project [Sum slices]), then run Analyze | Measure and extract the mass center (MC) coordinates (MX and MY) from the results windows.
      2. Apply the same algorithm mentioned before changing Organelle for MC. Thus, the angle (α) is defined by CC-MC (a) and CC-BC (b).
      3. Calculate the polarity using the following formula:
         
   3. Estimate lysosome recruitment to the synaptic area (Figure 2B).
      NOTE: This algorithm is used to quantify an organelle adjacent to the IS.
      1. Once the bead and cell areas have been determined, determine the angle of the vector between the CC and BC and then rotate the image to achieve a 0° angle.
      2. Set the fluorescent channel and project the image into one z-stack (Image | Stacks | Z-Project [Sum slices]), eliminate all the fluorescence that falls outside the cell and bead area together (run edit | clear outside in ImageJ).
      3. Using the rectangle tool selection, draw a rectangle adjacent to the bead and measure the synaptic area fluorescence (SAF). This rectangle is a quarter of the cell width.
      4. Select the entire image and run Analyze | Measure to obtain the whole cell fluorescence (WCF).
      5. Calculate the organelle fluorescence percentage adjacent to the IS using the following formula:
         
   4. Quantify recruitment of cellular components to the centrosome.
      NOTE: This algorithm, adapted from Obino et al.⁵, is used to quantify the enrichment of organelles at the centrosome area. Briefly, we consider the centrosome area as the domain in which the centrosome-associated organelle fluorescence remains constant or above 70% in the fluorescence/radius plot. It is essential to set this parameter at resting conditions because this radius could change upon activation.
      1. Once bead and cell areas have been determined, define the localization of the centrosome using the point tool selection (Figure 3B).
      2. First, determine the maximum area around the centrosome that is possible to quantify, by drawing a 3 µm-radius circle surrounding the centrosome.
      3. Use the ImageJ plugin Radial Profile, which measures the fluorescence in concentric circles and displays a fluorescence/radius plot.
      4. Identify the maximum radius within which at least 70% of the fluorescence intensity is maintained.
      5. Calculate the fluorescence density ratio using the following formula:
           = 
         NOTE: The fluorescence density ratio indicates the concentration of an organelle at the centrosome compared to its distribution in the whole cell.
2. Analysis of cell spreading and distribution of organelles in B cells activated on Ag-coverslips
   1. Estimate organelle distribution at the IS (Figure 4B).
      NOTE: This algorithm allows the quantification of the XY distribution of organelles and their concentration at the center of the IS. We define a ratio of fluorescence density between the central and the total synaptic area. The values obtained can vary from negative to positive, indicating a peripheral or a central distribution of the organelle, respectively.
      1. Determine the slice in which the cell is in contact with the cover.
         NOTE: To delimit the cell boundaries one can label the plasma membrane or actin which is enriched in the periphery of the IS.
      2. Change the type of the slice to 8-bit, then binarize (Process | Binary | Make Binary) and connect the nearest outsider points Process | Binary | Outline. Delimit the boundaries of the cell area (CA) using the polygon tool selection.
         NOTE: This step is useful to increase the contrast of the cell boundaries and make it easier to identify. Also, at this point, it is possible to apply the Analyze particle plugin of ImageJ to determine the CA automatically. However, other cells in the same field can interfere with the results.
      3. Take CA parameters (height and width) and delimit a central rounded area, which is separated from the boundaries by a quarter of the height and width values, consider this area as the Cell Center Area ( CCA).
      4. Calculate the fluorescence density distribution at the center of the IS using the following formula:
         
   2. Measure organelle distribution in Z planes (Figure 4C).
      NOTE: This analysis determines the general distribution of organelle fluorescence across Z planes of B cells activated onto Ag-coverslips, showing the percentage of fluorescence per Z fraction.
      1. To quantify the fluorescence distribution in Z, first determine the plane of the IS where the cell is in contact with the Ag-coverslip and then the plane corresponding to the upper limit of the cell.
      2. Draw a line across the cell center.
      3. Reslice the image in Z (Image | Stacks | Reslice), to obtain an XZ image.
      4. Measure the height and divide the XZ image into 10 consecutive rectangles of the same height (Z fraction) from the bottom (IS interface) to the top (upper side of the cell) and quantify the fluorescence signal in each one.
      5. Normalize the fluorescence intensity of each Z fraction by the sum of the total fluorescence of the 10 fractions.
      6. Plot the percentage of fluorescence intensity per Z fraction of the cell.

5. Isolation of centrosome-enriched fraction from resting and activated B cells

NOTE: Keep all solutions at 4 °C during the experiment to avoid protein degradation. This protocol was adapted from previous work^17,18.

1. Activate 2 x 10⁷ B cells with Ag-coated beads in 2 mL of CLICK medium + 2% heat-inactivated FBS (ratio 1:1). Consider non-activated B cells as resting B cells.
2. Add cytochalasin D (2 µM) and nocodazole (0.2 µM) and incubate for 1 h at 37 °C.
   NOTE: These drugs are used to gently detach the centrosome from the nucleus, by depolymerizing actin cytoskeleton and microtubules, respectively, to avoid nuclear contamination.
3. Wash each sample with 5 mL of cold 1x TBS (50 mM Tris-HCl, pH 7.6, 150 mM NaCl) and then with 1 mL of 0.1x TBS supplemented with 8% sucrose.
4. Resuspend the cells with 150 µL of centrosome lysis buffer (1 mM HEPES, pH 7.2, 0.5% NP-40, 0.5 mM MgCl₂, 0.1% beta-Mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF) or protease inhibitor cocktail) and pipette up and down until viscosity decreases, which indicates cell lysis is identified in the sample. Put the sample in a 1.5 mL tube.
5. Centrifuge at 10,000 x g for 10 min at 4 °C, to separate the organelles from the nucleus.
6. Carefully recover the supernatant and place it on top of a 1.5 mL tube with 300 µL of gradient buffer (GB) (10 mM PIPES pH 7.2, 0.1% Triton X-100, 0.1% beta-mercaptoethanol) containing 60% sucrose.
7. Centrifuge at 10,000 x g for 30 min at 4 °C to concentrate the centrosomes in the 60% sucrose fraction.
8. Meanwhile, prepare a discontinuous gradient in 2 mL ultracentrifuge tubes by overlaying 450 µL of GB + 70% sucrose with 270 µL of GB + 50% sucrose and then 270 µL of GB + 40% sucrose.
9. After the first centrifugation (centrosome-concentrated fraction), discard the upper fraction (less dense portion) until reaching the interface and vortex the remaining sample in the tube. Then, overlay on top of the discontinuous gradient previously prepared with the centrosome enriched sample.
   NOTE: Be careful not to disrupt the gradient, all pipetting procedures must be carefully performed.
10. Centrifuge at 40,000 x g for 1 h at 4 °C with minimal acceleration and with the centrifuge brake set to off, to avoid disrupting the gradient.
11. Collect 12 fractions of 100 µL into separate tubes beginning from the top.
12. Identify the centrosome enriched fractions by immunoblot using γ-tubulin as the centrosome marker.
    NOTE: We usually find centrosome-enriched extracts between fractions 6 and 8.