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

The protocol describes the isolation of lymphoid cells from human patient material and therefore requires an ethical approval. The work done in the present study was granted by the Uppsala Ethical Review Board (Dnr. 2013/387).

1. Isolation of Mononuclear Cells (MNCs) from Human Palatine Tonsils

CAUTION: All unscreened material of human origin like blood, tissues or body fluids should be regarded as potentially infected material. Therefore, recommended biosafety practices for handling the human tissues should be followed.

Note: To avoid contamination, all solutions and cell culture equipment must be sterile. All buffers and solutions should be pre-cooled and kept on ice. Tonsil tissues and isolated cells should be kept and handled on ice.

1. Obtain fresh tonsils from patients undergoing tonsillectomy or tonsillotomy (Figure 2). Plunge the tissue clumps into a 50 ml centrifuge tube filled with ice-cold sterile Hanks balanced salt solution (HBSS) supplemented with 5% fetal bovine serum (FBS), 10 mM Glutamine, 0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix (Penicillin, Streptomycin and Amphotericin B).
2. Keep the tubes with the submerged tonsil tissue samples at all times on ice. Try to process the tonsils as soon as possible, and no later than 3 hr after surgical removal.
3. Place the tonsil on a 60 mm plastic cell culture plate on ice and keep the tissue moistened with HBSS. Remove visible blood clots, fatty and connective tissues with a clean forceps from the tonsil surface.
4. Use a new 60 mm cell culture plate containing 5 ml HBSS. Cut the tonsil tissue clump into 3-10 mm fragments using a sterile pair of scissors and/or a scalpel.
5. Prepare a new 60 mm cell culture plate containing 10 ml of HBSS and place a 100 µm plastic cell strainer in the HBSS solution.
6. Transfer the dissected tonsil fragments into the cell strainer with a sterile forceps. Using the plunger end of a plastic syringe, smoothly squeeze the tissue fragments through the cell strainer. Make sure that the tonsil fragments are totally immersed in the HBSS.
7. Discard the cell strainer with the tissue remains and transfer the resulting cell suspension from the 60 mm cell culture plate into a 50 ml plastic centrifuge tube. Leave the tube on ice while proceeding with steps 1.8 and 1.9.
8. In the case of big tonsils, larger than 2 cm in size (Figure 2), squeeze the tonsil fragments in two cell strainers to avoid clogging of the mesh in the strainer.
9. Obtain the final volume of 35 ml of the cell suspension.
   Note: At this step it is possible to prepare the cell suspension for cryopreservation (see section 2).
10. Add 10 ml of density gradient solution such as Ficoll into a new 50 ml centrifuge tube. Gently overlay the cell suspension (step 1.9) on top of the density gradient solution. Avoid mixing of the cell suspension and the density gradient solution.
    Note: The density gradient solution has to be warmed up to RT.
11. Centrifuge the cell suspension in a swing-out rotor at 700 x g for 20 min at RT. Do not activate the brake function on the centrifuge as fast braking may disrupt the gradient. Also, use the lowest acceleration function available on the centrifuge.
    Note: After this centrifugation step, observe MNCs as a fluffy white layer at the interface, and red blood cells (RBCs), fibroblasts and cell debris as the sediment at the bottom of the tube.
12. Carefully collect the MNCs layer by using a 10 ml pipet. Place the cell suspension into a new 50 ml centrifuge tube.
13. Add 25 ml of ice-cold PBS to the cell suspension and spin the tube at 300 x g for 5 min in a swing-out rotor at 4 °C.
14. Remove the supernatant using a 25 ml pipet and repeat cell pellet washing step two more times. Finally resuspend the cell pellet in 15 ml of PBS.
    Note: After the final washing step, it is possible to cryopreserve the cell suspension (see section 2).
15. Apply 10 µl of cell suspension into hemocytometer cell counting chamber and count the cell number under a microscope. Dispense the appropriate amount of cells (e.g., 2-3 x 10⁷) in a 15 ml centrifuge tube for subsequent magnetic bead separation. Keep this aliquot on ice until step 3.2.

2. Cryopreservation of Tonsillar Cells

1. Prepare freezing medium (90% FBS and 10% DMSO) and keep it on ice. For long-term storage keep the freezing media at -20 °C.
2. Determine the total number of cells using a hemocytometer cell counting chamber (step 1.15). Calculate the required amount of freezing medium according to the desired frozen cell density (e.g., 10⁷ cells/ml of freezing medium).
3. Centrifuge the cell suspension at 300 x g for 5 min. Decant the supernatant without disturbing the cell pellet and resuspend the cell pellet in ice-cold freezing medium (from step 2.1).
4. Dispense 1 ml aliquots of the cell suspension into sterile vials designed for long-term storage in liquid nitrogen. Freeze the vials in an isopropanol chamber and store them at –80 °C O/N. For long-term preservation transfer the vials into a liquid nitrogen containing storage tank or a –140 °C cell freezer.
5. To thaw vials with frozen cells, warm them rapidly in a 37 °C water bath. Immediately when thawed, disperse the cell suspension into 10 ml of pre-warmed HBSS (with supplements, step 1.1) in a 15 ml centrifuge tube. Spin the tube at 300 x g for 5 min, remove HBSS and resuspend the cell pellet at the desired cell density in HBSS (with supplements, step 1.1).
   Note: The viability of the MNCs after thawing is of importance, since the presence of dead cells will decrease the final yield of purified B and T lymphocytes.
   Note: According to our experience the cell viability less then 80% will reduce the cell isolation efficiency.

3. Positive Selection of T Lymphocyte Population from Tonsillar MNCs

Note 1: This protocol is based on positive selection of human CD3⁺ T lymphocytes from tonsillar MNCs using magnetic beads coupled to the CD3 antibody. It is possible to start this section from fresh (Section 1) or the frozen (Section 2) MNCs.

Note 2: Start with 3 x 10⁷ MNCs. Do not exceed this cell number, since the separation columns may clog and this will reduce the isolation efficiency. Use bigger columns if more cells will be handled. The volumes used in this protocol have been experimentally optimized for the number of cells used in our experimental setup.

1. Prepare the separation buffer [PBS (pH 7.2), 0.5% bovine serum albumin (BSA) and 2 mM EDTA]. Filter the separation buffer through a 0.45 µm filter and store at 4 °C.
2. Spin the MNCs suspension (step 1.14) at 300 x g for 5 min at 4 °C. Resuspend the resulting cell pellet in 240 µl (80 µl per 10⁷ cells) of ice-cold separation buffer. Transfer the cell suspension into a sterile 2 ml tube.
3. Add 20 µl of CD3 magnetic antibody to the cell solution. Incubate the tubes for 1 hr at 4 °C with continuous gentle mixing to keep cells in suspension.
4. Transfer all of the cell suspension into a 15 ml tube containing 5 ml ice-cold separation buffer to wash the cells.
5. Centrifuge the tube at 300 x g for 10 min at 4 °C in a swing-out rotor.
6. Meanwhile set up the magnetic separator and column. Attach the magnetic separator to the stand and place the column in the separator. Wash the column by applying 500 µl of ice-cold separation buffer. Discard the flow through.
7. Place a new 15 ml collection tube below the column. Keep the collection tube on ice.
8. Discard the supernatant (step 3.5) by pipet and gently resuspend the cell pellet in 500 μl separation buffer. Apply the cell suspension on top of the pre-washed column and let it run through.
   Note: Cell clumps can clog the column and thus reduce the flow rate. To prevent this problem, place a 40 µm plastic cell strainer on top of the column. Passing the cells through this mesh will highly improve the efficiency of this method.
9. Wash the column 4 times with 1.5 ml of separation buffer (500 µl for 10⁷ cells). Wait for the column reservoir to be empty (no liquid should be observed in the column) before applying the next washing step.
   Note: Obtain the CD3 unlabeled cells, considered as B lymphocyte fraction, as they are eluted into the collection tube.
10. Remove the column from the separator and place into a new 15 ml collection tube. Pipette 2 ml of separation buffer onto the column. Using the plunger supplied with the column elute the positively selected T lymphocytes, which are regarded as the T lymphocyte fraction.
11. Determine the number of purified cells (step 1.15) if necessary. Cell suspensions are now ready for downstream experiments.

4. Flow Cytometry Analysis of Isolated Tonsillar B and T Lymphocytes

CAUTION: Paraformaldehyde solution is irritant and suspected carcinogen. Wear suitable protective clothing, gloves, and eye/face protection.

Note 1: This protocol describes the method for direct staining of the isolated B and T cells by Fluorescence Activated Cell Sorting (FACS) analysis. The main purpose of this step is to assess the purity of the isolated cell populations after CD3 magnetic antibody separation. For this purpose established B and T cell markers, fluorophore-conjugated monoclonal antibodies CD20-FITC and CD2-APC, are used. Also the inclusion of isotype control antibodies is highly recommended to distinguish the non-specific “background” binding of the CD2 and CD20 antibodies (see step 4.8).

Note 2: It is possible to keep the purified cell fractions in a 1% paraformaldehyde (PFA) solution in the dark at 4 °C until the time of staining (e.g., next day). Also the same procedure is applicable if there is a time gap between staining and FACS analysis. Remember that in both cases cells should be washed properly with PBSA (PBS containing 0.2% BSA) buffer, prior to staining or FACS analysis.

1. Take out 6 x 10⁶ cells of MNCs from step 1.15 (prior to applying to the column), B lymphocyte fraction from step 3.9 and T lymphocyte fraction from step 3.10.
2. Spin the cell suspensions at 300 x g for 5 min at 4 °C using a swing-out rotor. 
3. Remove the supernatant with pipet and wash the cell pellet once with 1 ml of ice-cold PBSA. Spin the cell suspensions at 300 x g for 5 min at 4 °C. Remove the supernatant with pipet and resuspend the cells in 600 µl of ice-cold PBSA buffer.
4. Add 100 µl of the cell suspension to the bottom of a FACS tube.
5. Add 100 µl of PBS containing 10% heat inactivated human serum to each tube, mix well and incubate for ~1 min at RT or 20 min on ice.
   Note: B lymphocytes carry Fc receptors. In order to block the Fc receptors the purified cell fractions are incubated with 10% heat inactivated human serum. The human serum is heat inactivated by incubation at 56 °C for 1 hr. Divide the heat inactivated serum into small aliquots and store frozen at -20 °C.
6. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C in a swing-out rotor.
7. Remove the supernatant with pipet and wash the cell pellet with 1 ml PBSA. Repeat the centrifugation (300 x g for 5 min at 4 °C).
8. Add 100 µl PBSA into each tube on ice. Add the appropriate amount of fluorophore-conjugated monoclonal antibody, according to the manufacturer’s recommendation (e.g., 20 µl of anti-CD20 and/or 5 µl of anti-CD2 antibodies in 100 µl of PBSA). Note: Do not forget to assign enough control tubes for the FACS analysis. In this protocol the following controls should be included: 1) cells stained with the anti-CD2 and anti-CD20 antibodies individually, 2) cells stained with the anti-CD2 and anti-CD20 isotype control antibodies individually and, 3) cells without addition of antibodies.
9. Briefly vortex the tube and incubate for 30 min at 4 °C in the dark.
10. Wash 2 times with PBSA. Add 2 ml of 1% PFA solution to the samples. Let the cells remain in the PFA solution at 4 °C in dark till the time of FACS analysis.
11. Immediately before running the samples in the FACS machine, wash the cells 2 times with PBSA (300 x g for 5 min at 4 °C). Resuspend samples in 1 ml of PBS and keep at 4 °C (or on ice), protected from light, prior to the separation on the flow cytometer.
12. Do the FACS sorting following the protocol from the flow cytometer manufacturer.

5. PCR Detection of Adenovirus DNA in Isolated Tonsillar B and T Lymphocytes

Note 1: The adenovirus hexon gene primers are AdRJC1 (5’-GACATGACTTTCGAGGTCGATCCCATGGA-3’) and AdRJC2 (5’-CCGGCTGAGAAGGGTGTGCGCAGGTA-3’)¹¹. These primers generate an amplicon of 139 bp. The host 18S rRNA gene can be detected with primers tp206 (5'-CCCCTCGATGCTCTTAGCTG-3’) and tp207 (5'-TCGTCTTCGAACCTCCGACT-3’). The expected amplicon size is 300 bp.

Note 2: Every PCR run includes a negative control (distilled H₂O) and a positive DNA control obtained from human B cell line (BJAB) infected with human adenovirus type 5¹².

1. Extract DNA (from at least 1 x 10⁶ cells, step 3.11) using silica membrane based column purification method. Extract RNA using phenol and guanidine isothiocyanate solution¹³.
2. Add 100 ng DNA from B and T lymphocytes to a reaction mixture containing 1x HF buffer, 0.2 mM of deoxynucleotide triphosphate mix (dNTP), 0.25 µM of each primer and 1 U of High-Fidelity DNA polymerase in a total volume of 20 µl.
3. Perform PCR amplification under the following cycling conditions: 30 sec denaturation at 98 °C, followed by 30 cycles of 98 °C for 10 sec, 67 °C (hexon) or 58 °C (18S rRNA) for 30 sec and 72 °C for 30 sec. The PCR reaction was ended with a final extension step of 7 min at 72 °C.
4. Separate the resulting PCR amplification products on a 1.5% agarose gel in 1x TBE buffer and visualize the expected bands by staining with nucleic acid stain.