The overall goal of this simple yet effective procedure is to isolate and enrich for rare antigen-binding B cells from human peripheral blood using magnetic nanoparticles for their phenotypic and functional analysis. This method can help answer key questions in the field of immunology regarding the phenotype and biology of rare antigen-binding B cells both before and following in-vivo antigen encounter. The major advantage of this technique is that it obviates problems associated with geometry, particularly when one is using a styrene plate or a large bead.
And it obviates problems associated with flow rate when one is isolating cells by flow cytometry to isolate rare cells in quite large starting cell populations. Immediately after collecting 30 to 50 milliliters of whole blood into heparinized blood collection tubes, mix the blood at a 1:1 ratio with sterile room-temperature magnesium and calcium-free PBS. Next, layer the blood mixture onto 10 milliliters of room-temperature density gradient solution in a sterile 50 milliliter conical tube using slow consistent pipette trigger pressure.
Separate the cells by centrifugation. Discard the plasma and platelet-containing upper layer and use a sterile pipette to transfer the mononuclear cell container buffy coat into a new sterile 50 milliliter tube. Fill the tube with up to 50 milliliters of PBS for washing of the mononuclear cells by centrifugation.
Then, resuspend the pellet in 10 milliliters of fresh PBS for counting and resuspend the cells at a 1x10^7 cells per milliliter concentration in fresh PBS on ice. To stain the cells for magnetic bead-binding, first transfer 1-2x10^6 cell aliquots into five milliliter polystyrene tubes for each fluorescence compensation and Fluorescence Minus One control as appropriate. Next, collect the remainder of the cell sample by centrifugation and resuspend the pellet to a 3-6x10^7 cells per milliliter concentration in sterile filtered cold FACS buffer.
Split the cell solution equally into four FACS tubes on ice and add human Fcgamma receptor blocking reagant to these four experimental sample fractions. At the end of the nonspecific-binding blocking incubation, add the appropriate fluorescence conjugated cell-surface staining antibodies to the cells in all four sample fractions. Then, add the appropriate biotinylated antigen to fractions A and D followed by the addition of a sufficient volume of unlabeled antigen to fraction B such that the majority of the receptors with an affinity of at least 1x10^6 molar are blocked.
Incubate all of the samples for 30 minutes on ice followed by a 30 minute incubation of fraction B with the appropriate biotinylated antigen. At the end of the incubations, wash the cells two times in one milliliter of ice cold FACS buffer per 5x10^7 cells. And resuspend the pellets in one milliliter of 2%formaldehyde per 5x10^7 cells for five minutes in the dark on ice.
After fixation, wash cells two more times as just demonstrated. And resuspend the pellets in one milliliter of fresh ice cold FACS buffer per 5x10^7 cells. Then label the cells with one to two micrograms of the appropriate secondary Streptavidin Conjugated Antibody for 20 minutes in the dark on ice and wash the cells two more times.
For magnetic nanoparticle-based enrichment, resuspend the A, B, and C cell fractions in one milliliter of cold Separation Buffer per 5x10^7 cells and filter the cells through individual 40 microliter strainers to eliminate any clumps. Next, add the fluorescent dye nanoparticles at the appropriate concentration for 10 minutes in the dark at four degrees Celsius with rotation and wash the samples two times in one milliliter of cold Separation Buffer. Resuspend the pellets in one milliliter of cold Separation Buffer per 5x10^7 cells.
Then equilibrate three magnetic columns on their appropriately-sized magnets with three milliliters of Separation Buffer each, allowing the entire volume of Separation Buffer to run through each column into a waste container. Now place one labeled 15 milliliter conical tube under each column and add the magnetic particle labeled cells to their respective columns. When the entire volume of each cell fraction has passed through each column, add three milliliters of fresh ice cold Separation Buffer to the top of each column, collecting the effluents in the labeled tubes.
After the second wash has completely drained through the columns, place the tubes of negatively selected cells from each fraction on ice and transfer the columns into new 15 milliliter tubes for each fraction. Fill each column with approximately six milliliters of fresh Separation Buffer and immediately plunge the buffer through the columns to collect the magnetically-bound cells. Then collect both the positive and negative cell fractions by centrifugation and resuspend the pellets in the appropriate solution for the planned downstream analysis.
In these graphs, the frequency of tetanus-toxoid reactive B cells from an individual who was last vaccinated against tetanus more than a decade ago demonstrates the ability of this method to be used to enrich the tetanus-toxoid-binding cells approximately seven-fold with the purity of about 4%The addition of a 50-fold excess of unlabeled tetanus toxoid to the cells from a subject who was vaccinated about three years prior to the blood draw. Before biotinylated antigen is added, the tetanus-toxoid binding is decreased by 83%demonstrating the specificity of antigen binding. When the biotinylated antigen is omitted from the procedure, only 0.2%of the B cells still bind, presumably, reflecting the binding of some non-tetanus antigen in the absorbent.
These graphs demonstrate how the frequency of tetanus-toxoid-specific naive B cells from a blood sample drawn seven days after the booster vaccination of a healthy subject, decreases from 84%to 64%after the boost. While the percentage of memory and plasmablast populations increases from 16%to 36%and zero to 40%respectively. Further, representative ELISPOT data from a peripheral blood sample obtained seven days post-boost from a healthy subject illustrate the four-fold enrichment in tetanus-toxoid antibody-secreting cell frequency observed in the isolated tetanus-toxoid-binding B cells.
Once mastered, this technique can be completed in about three to four hours with all the proper controls. The method can be combined with secondary assays, for example, transfer of cells into adopted recipients for subsequent studies of cell fate and function. After watching this video, you should have a good understanding of how to isolate and enrich for rare antigen-binding B cells from human peripheral blood using the proper controls.
