The overall goal of this protocol is to demonstrate the preparation of a CD4+T cell suspension for staining to visualize GD3 and GD2 ganglioside localization on the cell surface using a low number of cells. This method visualization of membrane glycolipids in a low number of small suspension cells, as is the case of mononuclear cells. The main advantage of this technique is that you can evaluate ganglioside expression and localization in low amounts of CD4+T cells by optimizing sample use.
The implications of this technique could extend toward research and diagnosis of some types of T cell leukemia. As we have found, some leukemia cell lines upregulate GD3 and GD2 ganglioside expression. Begin by diluting two milliliters of peripheral blood from healthy human donors with two milliliters of sterile PBS EDTA.
Slowly dispense the diluted blood on top of three milliliters of 1.077 density sucrose solution. Then, centrifuge for 30 minutes at 250 times G at 21 degrees Celsius with no break to generate the gradient of peripheral blood mononuclear cells. After centrifugateion the PBMCs can be clearly observed as a white ring.
Collect the PBMCs with a pipette and transfer to a sterile tube for washing. After washing the cells with PBS EDTA, purify naive CD4+T cells by negative selection with magnetic beads using a commercially available kit. Once a population of greater than 95%purity CD4+T cells has been obtained, prepare 24 well cell culture plates by adding 250 microliters of five micrograms per milliliter anti-CD3 monoclonal antibody in PBS to each well.
Incubate the plate at 37 degrees Celsius for at least two hours. Following the incubation, remove the anti-CD3 antibody dilution from the plates and wash twice using sterile 1x PBS. Then, dilute the naive CD4+T cells with supplemented advanced RPMI 1640 medium to a density of one times 10 to the sixth cells per milliliter.
Next, add 500 microliters of diluted cells to the anti-CD3 coated wells of the 24 well plate. Then complete the stimulation of naive CD4+T cells in anti-CD3 coated wells by adding one microgram per milliliter anti-CD28 antibody. Incubate the resting and activated CD4+T cells for zero to 72 hours at 37 degrees Celsius and 5%carbon dioxide.
After the allotted incubation time has elapsed, collect the resting and activated naive CD4+T cells from the 24 well plates. Perform a cell count and if necessary, dilute the cells with fresh supplemented medium to a concentration of one times 10 to the sixth cells per milliliter. Seed 500 microliters of diluted cells into the wells of a 24 well plate containing poly-L-lysine coated cover slips or a slide chamber and incubate at 37 degrees Celsius and 5%carbon dioxide for a minimum of six hours.
Following the incubation, the CD4+T cells will be adhered to the cover slips. Carefully remove the medium from the wells and then slowly add 500 microliters of sterile PBS. PBS addition during washing should be done very carefully, especially with slide chambers with less than 50, 000 cells per well are being prepared.
Aspirate the PBS and add another 500 microliters of PBS to the wells to thoroughly remove the culture medium. After removing all of the PBS, add 500 microliters of filtered 4%paraformaldehyde and incubate for 30 minutes at room temperature to fix the cells. Following fixation, remove the fixative and wash at least twice with PBS.
If desired, the plate can be stored with 500 microliters of PBS in each well for several days at four degrees Celsius. First, remove the PBS from the fixed cells and add 500 microliters of cold blocking buffer. Incubate for 20 minutes on ice.
During incubation, it is critical not to vigorously shake the plate, as shaking will take off the cells. Carefully aspirate the blocking buffer and add blocking buffer containing 2.5 micrograms per milliliter of one of the primary antibodies or PE conjugated anti-CD25, APC conjugated anti-TCR, or an isotype control. Incubate for two hours on ice and protect from light or overnight at four degrees Celsius.
Following the incubation, carefully remove the antibody solution and slowly add 500 microliters of PBS. Then add the appropriate secondary antibodies diluted in blocking buffer to 0.1 micrograms per milliliter. Protect from light and incubate for one hour on ice while taking care to avoid shaking.
After washing three times in PBS as before, add Hoecht stain diluted in PBS to 0.1 micrograms per milliliter and incubate protected from light for 15 minutes at room temperature. Then place the washed slides on a paper towel and add 20 microliters of mounting solution or commercial mounting solution to prevent fading and quenching of fluorescence. Finally, pick up a cover slip with fine tweezers, turn it face down and place it on top of the mounting solution so that the side of the cover slip where cells are attached is in contact with the solution.
Seal the cover slips with nail polish and analyze using fluorescence or confocal microscopy. This image shows bright field visualization of fixed CD4+T cells 72 hours after activation. Validation of efficient activation after staining is assessed using anti-CD25 antibody shown in red.
The GD3 ganglioside is localized intracellularly and on the cell surface of activated CD4+T cells, as assessed by staining with anti-GD3 antibody shown in green. GD3, again shown in green, does not co-localize with the TCR, shown again in red, as assessed with anti-GD3 and anti-TCR antibody staining. However, the GD2 ganglioside does co-localize with the TCR and CD4+T cells, as assessed by staining with anti-GD2, shown in green, and anti-TCR antibody staining, shown in red.
The GD3 ganglioside staining shows the intracellular and cell surface localization in a subset of activated PBMCs. While the GD2 ganglioside staining shows intracellular and cell surface localization in all activated PBMCs. Following this procedure of adherence and manipulation of the CD4+T cells, other methods like intracellular compartment staining can be performed in order to answer additional questions like the specific intracellular localization or processing of gangliosides.
After watching this video, you should have a good understanding of how to handle and adhere a low number of CD4+T cells to obtain high quality fluorescent microscopy images to analyze ganglioside expression.
