Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

The overall goal of this procedure is to obtain BNT lymphocyte fractions from human tonsil samples. This method can help answering the key questions in the field of virology, like what are the roles of BT lymphocyte fractions in the pathogenesis of viral diseases Within three hours of their surgical removal. Transport the tonsil tissue to the lab in a 50 milliliter centrifuge tube filled with ice cold, sterile HBSS containing supplements.

Then using the appropriate biosafety procedures recommended for handling human tissue. Moisten the tissue with fresh supplement containing HBSS and transfer the tonsils to a 60 millimeter plastic cell culture plate on ice. Using forceps, remove any visible blood clots or fatty and connective tissues, and then transfer the tonsils into a new 60 millimeter plate containing five milliliters of HBSS with supplements.

Next, cut. The tonsil tissue clump into three to 10 millimeter fragments, followed by transfer of the fragments into a 100 micrometer plastic cell strainer in a new 60 millimeter cell culture plate containing 10 milliliters of fresh HBSS with supplements using the plunger end of a plastic syringe smoothly. Squeeze the tissue fragments through the cell strainer, making sure that the tonsil fragments remain totally immersed in the HBSS when all of the tissue has been macerated, discard the strainer and transfer the resulting cell suspension into a 50 milliliter plastic centrifuge tube on ice.

Bring the cell solution to a final volume of 35 milliliters with fresh HBSS containing supplements, and then add 10 milliliters of room temperature density gradient solution into a few 50 milliliter conical tubes. Gently overlay the cell suspensions on top of the density gradients, taking care not to mix the solutions and then separate the cells by centrifugation. The mononuclear cells will separate into a fluffy white layer at the interface with the red blood cells fibroblasts and cell debris located in the sediment at the bottom of the tube.

Using a 10 milliliter pipette, carefully collect the mononuclear cell layer and pull the mononuclear cells in a new 50 milliliter tube. Then wash the cells three times in 25 milliliters of ice cold PBS per each wash and resuspend the final pellet in 15 milliliters of PBS. After counting the number of viable cells, place the cells on ice to isolate the tonsor T cells by magnetic bead separation.

Spin down the mononuclear cell suspension and resuspend the pellet in 80 microliters of ice cold separation buffer per one times 10 to the seven cells. Then transfer the cell suspension into a sterile two milliliter tube and incubate in 20 microliters of CD three magnetic antibody for one hour at four degrees Celsius with continuous gentle mixing to keep the cells and suspension at the end of the incubation, transfer the cells into a 15 milliliter tube containing five milliliters of ice cold separation buffer and wash them during the wash. Attach the magnetic separator to the stand and load a column onto the magnet.

Wash the column with 500 microliters of ice cold separation buffer, and then place a 40 micrometer strainer on top of the column. Now re suspend the cell pellet in 500 microliters of fresh separation buffer and load the cells through the strainer onto the column collecting the B-cell containing EIT in a 15 milliliter conical. Two bon ice when all of the cells have passed into the collection tube.

Wash the column four times with 500 microliters of separation buffer per one times 10 to the seven cells waiting for the column reservoir to be empty before applying the next wash. After the last wash, transfer the column into a new 15 milliliter collection tube. Then using the column plunger to press two milliliters of separation buffer through the column into the tube to collect the CD three positive T cell fraction.

To analyze the isolated B and t tonsor cell fractions, pellet the CD three positive and CD three negative cell fractions and wash both sets of cells in one milliliter of PBS supplemented with 0.2%BSA or PBSA after the second spin. Distribute 100 microliter aliquots of the cells and PBSA into the appropriate number of tubes for fax labeling, and add the appropriate amount of fluoro four conjugated monoclonal or control antibody to each tube. Then briefly vortex the tubes and incubate them for 30 minutes at four degrees Celsius in the dark at the end of the incubation.

Wash the tubes two times with fresh PBSA and then fix the cell staining with 1%paraform aldehyde to detect the presence of adenovirus DNA in the cells by PCR. Perform the PCR amplification under the appropriate cycling conditions, separating the resulting PCR amplification products on a 1.5%AGROS gel and interestate EDTA buffer. Finally, visualize the resulting bands with the appropriate nucleic acid stain.

According to standard protocols, a successful separation of the tonsor mononuclear cells results in highly purified subpopulations of BNT lymphocytes as confirmed by flow cytometric analysis using anti CD 20 and anti CD two antibodies to detect the b and t lymphocyte populations respectively. In contrast, an inefficient separation of the mononuclear cells results in cell fractions that are a mixture of cells from both the b and t lymphocytes. Subpopulations, DNA and RNA can be extracted from the magnetic bead separated b and t lymphocytes for downstream analysis as just demonstrated.

For example, here, the specific detection of adenovirus DNA in TONSOR T lymphocytes as detected by PCR is shown quality cytoplasmic. RNA can be extracted from isolated tonsor B NT lymphocytes as well. Once monster two tonsil samples can be processed within six hours if the technique is performed properly.

While attempting this procedure, it's important to process the tonsil samples as fast as possible. The number of the washing steps and the volume of the washing buffer are also critical parameters for a good result. Following this procedure, other methods like QPCR and R-T-Q-P-C-R could be performed to answer additional questions about the quantitative presence of viral DNA and mRNA after its development.

This technique paved the way for the researchers in the virology field to explore the pathogenesis and latency of different viral species in the human lymphoid tissues. After watching this video, you should have a good understanding of how to isolate BT lymphocyte fractions from human tonsil samples. Don't forget that working with unscreened materials of human origin like blood and tissues could be extremely hazardous.

Therefore, appropriate by safety practices should be followed.