This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgstr&#246;ms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

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,&#160;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.
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare a new 60 mm cell culture plate containing 10 ml of HBSS and place a 100 &#181;m plastic cell strainer in the HBSS solution.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Carefully collect the MNCs layer by using a 10 ml pipet. Place the cell suspension into a new 50 ml centrifuge tube.</li>
	<li>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 &#176;C.</li>
	<li>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).</li>
	<li>Apply 10 &#181;l 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.</li>
</ol>
2. Cryopreservation of Tonsillar Cells
<ol>
	<li>Prepare freezing medium (90% FBS and 10% DMSO) and keep it on ice. For long-term storage keep the freezing media at -20 &#176;C.</li>
	<li>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).</li>
	<li>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).</li>
	<li>Dispense 1 ml aliquots of the cell suspension into sterile vials designed for long-term storage in liquid nitrogen.&#160;Freeze the vials in an isopropanol chamber and store them at &#8211;80 &#176;C O/N. For long-term preservation transfer the vials into a liquid nitrogen containing storage tank or a &#8211;140 &#176;C cell freezer.</li>
	<li>To thaw vials with frozen cells, warm them rapidly in a 37 &#176;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.</li>
</ol>
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.&#160;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.
<ol>
	<li>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 &#181;m filter and store at 4 &#176;C.</li>
	<li>Spin the MNCs suspension (step 1.14) at 300 x g for 5 min at 4 &#176;C. Resuspend the resulting cell pellet in 240 &#181;l (80 &#181;l per 107 cells) of ice-cold separation buffer. Transfer the cell suspension into a sterile 2 ml tube.</li>
	<li>Add 20 &#181;l of CD3 magnetic antibody to the cell solution. Incubate the tubes for 1 hr at 4 &#176;C with continuous gentle mixing to keep cells in suspension.</li>
	<li>Transfer all of the cell suspension into a 15 ml tube containing 5 ml ice-cold separation buffer to wash the cells.</li>
	<li>Centrifuge the tube at 300 x g for 10 min at 4 &#176;C in a swing-out rotor.</li>
	<li>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 &#181;l of ice-cold separation buffer. Discard the flow through.</li>
	<li>Place a new 15 ml collection tube below the column. Keep the collection tube on ice.</li>
	<li>Discard the supernatant (step 3.5) by pipet and gently resuspend the cell pellet in 500 &#956;l 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 &#181;m plastic cell strainer on top of the column. Passing the cells through this mesh will highly improve the efficiency of this method.</li>
	<li>Wash the column 4 times with 1.5 ml of separation buffer (500 &#181;l 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.</li>
	<li>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.</li>
	<li>Determine the number of purified cells (step 1.15) if necessary. Cell suspensions are now ready for downstream experiments.</li>
</ol>
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 &#8220;background&#8221; 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 &#176;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.
<ol>
	<li>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.</li>
	<li>Spin the cell suspensions at 300 x g for 5 min at 4 &#176;C using a swing-out rotor.&#160;</li>
	<li>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 &#176;C. Remove the supernatant with pipet and resuspend the cells in 600 &#181;l of ice-cold PBSA buffer.</li>
	<li>Add 100 &#181;l of the cell suspension to the bottom of a FACS tube.</li>
	<li>Add 100 &#181;l 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 &#176;C for 1 hr. Divide the heat inactivated serum into small aliquots and store frozen at -20 &#176;C.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min at 4 &#176;C in a swing-out rotor.</li>
	<li>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 &#176;C).</li>
	<li>Add 100 &#181;l PBSA into each tube on ice. Add the appropriate amount of fluorophore-conjugated monoclonal antibody, according to the manufacturer&#8217;s recommendation (e.g., 20 &#181;l of anti-CD20 and/or 5 &#181;l of anti-CD2 antibodies in 100 &#181;l 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.</li>
	<li>Briefly vortex the tube and incubate for 30 min at 4 &#176;C in the dark.</li>
	<li>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 &#176;C in dark till the time of FACS analysis.</li>
	<li>Immediately before running the samples in the FACS machine, wash the cells 2 times with PBSA (300 x g for 5 min at 4 &#176;C). Resuspend samples in 1 ml of PBS and keep at 4 &#176;C (or on ice), protected from light, prior to the separation on the flow cytometer.</li>
	<li>Do the FACS sorting following the protocol from the flow cytometer manufacturer.</li>
</ol>
5. PCR Detection of Adenovirus DNA in Isolated Tonsillar B and T Lymphocytes
Note 1: The adenovirus hexon gene primers are AdRJC1 (5&#8217;-GACATGACTTTCGAGGTCGATCCCATGGA-3&#8217;) and AdRJC2 (5&#8217;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#8217;)11. These primers generate an amplicon of 139 bp. The host 18S rRNA gene can be detected with primers tp206 (5&#39;-CCCCTCGATGCTCTTAGCTG-3&#8217;) and tp207 (5&#39;-TCGTCTTCGAACCTCCGACT-3&#8217;). 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.
<ol>
	<li>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.</li>
	<li>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 &#181;M of each primer and 1 U of High-Fidelity DNA polymerase in a total volume of 20 &#181;l.</li>
	<li>Perform PCR amplification under the following cycling conditions: 30 sec denaturation at 98 &#176;C, followed by 30 cycles of 98 &#176;C for 10 sec, 67 &#176;C (hexon) or 58 &#176;C (18S rRNA) for 30 sec and 72 &#176;C for 30 sec. The PCR reaction was ended with a final extension step of 7 min at 72 &#176;C.</li>
	<li>Separate the resulting PCR amplification products on a 1.5% agarose gel in 1x TBE buffer&#160;and visualize the expected bands by staining with nucleic acid stain.</li>
</ol>

<ol><li>Nave, H., Gebert, A., Pabst, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphology+and+immunology+of+the+human+palatine+tonsil%5BTitle%5D)+AND+%22Anat+Embryol+%28Berl%29%22%5BJournal%5D)">Morphology and immunology of the human palatine tonsil.</a> Anat Embryol (Berl). 204, 367-373 (2001).</li>
<li>Perry, M., Whyte, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunology+of+the+tonsils%5BTitle%5D)+AND+%22Immunology+today%22%5BJournal%5D)">Immunology of the tonsils.</a> Immunology today. 19, 414-421 (1998).</li>
<li>Alkhalaf, M. A., Guiver, M., Cooper, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+and+quantitation+of+adenovirus+DNA+from+human+tonsil+and+adenoid+tissues%5BTitle%5D)+AND+%22J+Med+Virol%22%5BJournal%5D)">Prevalence and quantitation of adenovirus DNA from human tonsil and adenoid tissues.</a> J Med Virol. 85, 1947-1954 (2013).</li>
<li>Brandtzaeg, P., Halstensen, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunology+and+immunopathology+of+tonsils%5BTitle%5D)+AND+%22Adv+Otorhinolaryngol%22%5BJournal%5D)">Immunology and immunopathology of tonsils.</a> Adv Otorhinolaryngol. 47, 64-75 (1992).</li>
<li>Imai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epstein-Barr+virus+%28EBV%29-carrying+and+-expressing+T-cell+lines+established+from+severe+chronic+active+EBV+infection%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection.</a> Blood. 87, 1446-1457 (1996).</li>
<li>Veen, J., Lambriex, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+of+adenovirus+to+lymphocytes+in+naturally+infected+human+tonsils+and+adenoids%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Relationship of adenovirus to lymphocytes in naturally infected human tonsils and adenoids.</a> Infect Immun. 7, 604-609 (1973).</li>
<li>Friedman, M., Wilson, M., Lin, H. C., Chang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Updated+systematic+review+of+tonsillectomy+and+adenoidectomy+for+treatment+of+pediatric+obstructive+sleep+apnea%2Fhypopnea+syndrome%5BTitle%5D)+AND+%22Otolaryngol+Head+Neck+Surg%22%5BJournal%5D)">Updated systematic review of tonsillectomy and adenoidectomy for treatment of pediatric obstructive sleep apnea/hypopnea syndrome.</a> Otolaryngol Head Neck Surg. 140, 800-808 (2009).</li>
<li>Garnett, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Latent+species+C+adenoviruses+in+human+tonsil+tissues%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Latent species C adenoviruses in human tonsil tissues.</a> J Virol. 83, 2417-2428 (2009).</li>
<li>Garnett, C. T., Erdman, D., Xu, W., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+and+quantitation+of+species+C+adenovirus+DNA+in+human+mucosal+lymphocytes%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes.</a> J Virol. 76, 10608-10616 (2002).</li>
<li>Perez, M. E., Billordo, L. A., Baz, P., Fainboim, L., Arana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+memory+B+cells+isolated+from+blood+and+tonsils+are+functionally+distinctive%5BTitle%5D)+AND+%22Immunol+Cell+Biol%22%5BJournal%5D)">Human memory B cells isolated from blood and tonsils are functionally distinctive.</a> Immunol Cell Biol. 92, 882-887 (2014).</li>
<li>Cooper, R. J., Yeo, A. C., Bailey, A. S., Tullo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenovirus+polymerase+chain+reaction+assay+for+rapid+diagnosis+of+conjunctivitis%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.</a> Invest Ophthalmol Vis Sci. 40, 90-95 (1999).</li>
<li>Zhang, Y., Huang, W., Ornelles, D. A., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+adenovirus+latency+in+human+lymphocyte+cell+lines%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Modeling adenovirus latency in human lymphocyte cell lines.</a> J Virol. 84, 8799-8810 (2010).</li>
<li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction%5BTitle%5D)+AND+%22Anal.+biochem%22%5BJournal%5D)">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal. biochem. 162, 156-159 (1987).</li>
<li>Watanabe, T., Yoshizaki, K., Yagura, T., Yamamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+antibody+formation+by+human+tonsil+lymphocytes%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">In vitro antibody formation by human tonsil lymphocytes.</a> J Immunol. 113, 608-616 (1974).</li>
</ol>

The authors have nothing to disclose.

One of the most important factors affecting the outcome of this protocol is the use of fresh tonsillar material as the starting material. Therefore, the tonsil samples should be processed within 3 hr after surgery. Tonsils could be obtained from both adults and children. The tonsillar material from the children is usually smaller due to the partial surgical removal of the tonsils (tonsillotomy). Therefore, the number of MNCs obtained from tonsillotomy samples (1 x 107-1 x 108) is less compared to to...

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called &#8220;Waldeyer&#180;s ring&#8221;. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.
The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2. 
Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed. 
Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

<table><tbody><tr><td>Hanks balanced salt solution (HBSS)&amp;nbsp;</td><td>Gibco</td><td>14175-053</td><td>Contains 5% fetal bovine serum (FBS), 10 mM Glutamine,&amp;nbsp; &amp;nbsp;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td></tr><tr><td>Freezing medium</td><td></td><td></td><td>90% FBS and 10% DMSO</td></tr><tr><td>MACS buffer</td><td></td><td></td><td>PBS (pH 7.2), 0.5% BSA and 2mM EDTA</td></tr><tr><td>PBSA&amp;nbsp;</td><td></td><td></td><td>PBS containing 0.2% BSA</td></tr><tr><td>PFA</td><td></td><td></td><td>PBS containing 1% paraformaldehyde (PFA). Make fresh. PFA is suspected carcinogen. Wear gloves and goggles.</td></tr><tr><td>Antibiotic-Antimycotic mix</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>60 mm Petridish</td><td>Nunc</td><td>150326</td><td></td></tr><tr><td>Dissecting foreceps</td><td>Fisher Scientific</td><td>1381241</td><td></td></tr><tr><td>Straight iris scissors&amp;nbsp;</td><td>Fisher Scientific</td><td>12912055</td><td></td></tr><tr><td>disposable scalpels</td><td>Swann-Morton</td><td>REF 0501</td><td></td></tr><tr><td>100 &amp;mu;m plastic cell strainer</td><td>Corning Life Sciences</td><td>352360</td><td></td></tr><tr><td>40 &amp;mu;m plastic cell strainers&amp;nbsp;</td><td>Corning Life Sciences</td><td>352340</td><td></td></tr><tr><td>2 ml plastic syringe</td><td>BD Biosciences&amp;nbsp;</td><td>300185</td><td></td></tr><tr><td>15 ml conical centrifuge tubes</td><td>SARSTEDT</td><td>62554502</td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td>SARSTEDT</td><td>62547254</td><td></td></tr><tr><td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td><td>Thermo Scientific Heraeus Megafuge 16R</td><td></td><td></td></tr><tr><td>Ficoll&amp;ndash;Hypaque</td><td>Sigma-Aldrich</td><td>F5415-50ML</td><td>Ficoll solution</td></tr><tr><td>Fetal calf serum</td><td>Biological industries</td><td>040071A</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>SIGMA</td><td>D2650-5X5ML</td><td></td></tr><tr><td>MACS MS columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>MACS human CD3 MicroBeads</td><td>Miltenyi Biotec</td><td>130-092-881</td><td></td></tr><tr><td>MACS separator (Octo MACS)</td><td>Miltenyi Biotec</td><td>130-042-109</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Merck Millipore</td><td>1120180100</td><td></td></tr><tr><td>EDTA</td><td>AnalaR NORMAPUR</td><td>20302.293</td><td></td></tr><tr><td>CD2 conjugated to allophycocyanin (CD2-APC)&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>560642</td><td></td></tr><tr><td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>556632</td><td></td></tr><tr><td>Human serum&amp;nbsp;</td><td>Rockland Immunochemicals</td><td>D119-0100</td><td></td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Scientific</td><td>F-530L</td><td></td></tr><tr><td>FACS tubes</td><td>BD Falcon</td><td>352003</td><td></td></tr><tr><td>Cryotube</td><td>SARSTEDT</td><td>72379</td><td></td></tr><tr><td>BD LSRII flowcytometer</td><td>BD Biosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>BD FACSDiva 4.1 software</td><td>BD Biosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>Nucleospin Blood&amp;nbsp;</td><td>Macherey-Nagel</td><td>740951.50</td><td>DNA isolation kit</td></tr><tr><td>TRIzol&amp;nbsp; reagent</td><td>Life technologies</td><td>15596</td><td>RNA isolation reagent</td></tr><tr><td>Hemocytometer</td><td>The Paul Marienfeld GmbH &amp;amp; Co. KG</td><td>0610030</td><td>cell counter device</td></tr><tr><td>GelRed</td><td>Biotium</td><td>41003</td><td>Nucleic acid gel stain&amp;nbsp;</td></tr></tbody></table>

efficient isolation protocol for b and t lymphocytes from human palatine tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

An efficient separation of tonsillar MNCs results in highly purified subpopulations of B and T lymphocytes. This was confirmed by FACS analysis using anti-CD20 and anti-CD2 antibodies to detect B and T lymphocyte populations, respectively (Figure 3A). In contrast, an inefficient separation of MNCs results in cell fractions that are a mixture of cells from both B and T lymphocyte subpopulations (Figure 3B).
The isolated tonsillar B and T lymphocytes (Fi...

Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

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

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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15.8K Views.  Uppsala Biomedical Center, Uppsala University. Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Genetics

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biology

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

Cancer Research

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

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

Podsumowanie

Przeglądaj więcej filmów

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from Sézary Syndrome Patients for Transcriptomic Profiling

Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolation and Activation of Murine Lymphocytes

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue