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

Summary

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.

Abstract

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term “tonsils” 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.

Introduction

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 “tonsils” 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 “Waldeyer´s ring”. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract^1,2. Indeed, numerous reports have shown that both bact....

Protocol

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 e....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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