The purpose of the method is to generate an immunological synapse which is an example of cell-to-cell conjugation formed by an antigen-presenting cell and a T-helper lymphocyte. Our aim is to record the first stages of the immune synapse formation and to record the trafficking events occurring in the T-helper lymphocyte. These eventually will lead to polarized secretion at the immune synapse.
The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy, and subsequent image processing. This improves the signal-to-noise ratio of the images, enhances the temporal resolution, and allows the simultaneous acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is compatible with end point cell fixation protocols that will allow immunofluorescence staining and analysis.
This protocol is also compatible with laser scanning fluorescent microscopy and other state-of-the-art microscopy techniques. Demonstrating the procedure will be Ana Bello, a graduate student, Alejandro Garrido, a technician, and Solange Moreno, a graduate student. To begin this procedure, add 150 microliters of fibronectin to each well of an eight microwell chamber slide and incubate it at 37 degrees Celsius for 30 minutes to one hour.
After the incubation period, aspirate the fibronectin and wash each well with 200 microliters of PVS for two minutes with gentle shaking. Repeat this wash once more and leave the second wash in the well. Transfer 10 milliliters of a confluent preculture of Raji cells to a 15 milliliter V-bottom tube.
Centrifuge at 300 times G and at room temperature for five minutes. Discard the supernatant and gently resuspend the cell pellet in warm, complete medium at a concentration of 1 million cells per milliliter. To label the Raji cells, transfer the required number of cells in culture medium to a two milliliter tube.
For the eight microwell chamber slide, a total of 1.6 milliliters of cell suspension is needed. Add cell tracker blue to a final concentration of 10 micromolar. Resuspend the cell tracker blue-stained cells and transfer 200 microliters of the cell suspension into each well of the prepared fibronectin-coated chamber slides.
Incubate the chamber slide at 37 degrees Celsius with five percent carbon dioxide for 30 minutes to one hour. After this, gently shake the chamber slides on the microscope to ensure that the Raji cells are adhered. Wash each well carefully with warm, complete cell culture medium to eliminate excess cell tracker blue.
Ensure that the Raji cells are adhered to the bottom of the plates, and they display gaps among each other and they are not confluent. 50-60%of cell confluence is appropriate. First, add Staphylococcal Enterotoxin E at a concentration of one microgram per milliliter to each well.
Ensure that Raji cells are pulsed with superantigen otherwise the T-cell receptor from the Jurkat cells will not recognize the SCE and synapse will not be formed. Incubate the chamber slide at 37 degrees Celsius with five percent carbon dioxide for at least 30 minutes. Next, obtain a previously-growing culture of Jurkat cells at a concentration between 1 and 2 million cells per millimeter.
Observe the cells under a phase contrast microscope and then transfer the cells to a 15 milliliter V-bottom tube. Centrifuge at 300 times G and at room temperature for five minutes. Discard the supernatant and gently resuspend the cells in warm, complete culture medium at a concentration of 1 million cells per milliliter.
Then maintain the Jurkat cells in culture at 37 degrees Celsius with five percent carbon dioxide until ready to use. Retrieve the chamber slides containing the cell tracker blue-labeled Staphylococcus Enterotoxin E pulsed-adhered Raji cells from the incubator. Carefully aspirate the culture medium from each well one by one with a pipette placed in a corner of the well.
Immediately replace the medium with 200 microliters of the resuspended Jurkat cells in cell culture medium. If a time lapse is being performed, quickly proceed to the microscope. Locate the chambered slide on the microscope staging waiter and select some appropriate fields.
Prepare the microscope and incubation chamber prior to imaging. After the Jurkat cells have been added to each well containing the Raji cells, quickly locate the microwelled chamber slide on the preheated microscope staged incubator and select some XY positions. If only an endpoint experiment is planned, incubate the chamber slide at 37 degrees Celsius with five percent carbon dioxide for one to two hours.
After the culture period, check for conjugate formation and subsequently fix the conjugates as outlined in the text protocol. To fix the cells, add warm RPMI without FCS and gently shake. Aspirate the RMPI and add 200 microliters of either PFA or prechilled acetone to each well.
Incubate the chamber slide at room temperature or on ice for 20 minutes. Then wash each well twice with PVS and add 200 microliters of quenching solution. In this study, Jurkat-Raji immune synapse conjugates are generated and the early stages of immunological synapse formation are properly imaged.
This strategy induces the immunological synapse formation while simultaneously performing the time lapse imaging. Representative synaptic Jurkat-Raji conjugates obtained from this technique are shown here, including an image of the transmittance channel, the cell tracker blue channel, and both of these channels merged. Some synaptic conjugates can be seen, including those made up of only one Jurkat cell and several Raji cells, which are complex conjugates.
Decreasing cell concentrations will circumvent the formation of complex cellular conjugates but may not provide enough cell conjugates for the subsequent analysis of polarized traffic, which will in turn decrease chances to find and to image emerging synaptic conjugates. A deconvolution of the GFP-CD63 fluorescence channel images is performed with an appropriate deconvolution software using the wide field optical option and the proper optical parameters. This deconvoluted channel was subsequently merged to the cell tracker blue broad channel.
There is an improvement of both the signal-to-noise ratio and the sharpness. A representative Z stack of a fixed immunological synapse conjugate is shown here. Immunofluorescence is performed using phalloidin to visualize the F-actin, anti-CD63 to visualize the MVB, and anti-gamma-tubulin to visualize MTOC while cell tracker blue labeled the Raji cells.
The optional transfixing of the Jurkat cells will allow the visualization of the traffic of the secretory granules in living cells. For instance, when GFP-CD63 is expressed, the movement of the GFP-CD63-decorated vesicles can be recorded. The protocol is compatible with end point fixation protocols that will allow further immunofluorescence staining protocols and analyses.
Productiveness experimental models exist that may simplify imaging in the immune synapse but these models do not emulate the complex and irregular surface on the antigen-presenter cells that may produce non-physiological interactions at the immune synapse. The approach described here is suitable to reproduce and confirm some biological complexities occurring at the immune synapse. Superantigen is a dangerous toxin so please be sure to wear gloves, and drop the used tip on the hazardous box.
