Summary

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Published: November 16, 2015
doi:

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 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.

Protocol

该协议描述了从人类患者材料淋巴细胞的分离,因此,需要一个伦理批准。在本研究中所做的工作给予的乌普萨拉伦理审查委员会(DNR。三百八十七分之二千〇一十三)。 1.分离单个核人力腭扁桃体细胞(MNCs)的警告:人类起源如血液,组织或体液的所有非屏蔽材料应被视为可能受感染的材料。因此,处理的人体组织推荐的生物安全的做法应该得到遵守?…

Representative Results

的扁桃体跨国公司结果在B和T淋巴细胞的高度纯化的亚群有效分离。这是通过FACS分析使用抗CD20和抗-CD2抗体,以检测B和T淋巴细胞群,分别(图3A)证实。与此相反,跨国公司导致细胞级分是从两个B和T细胞亚群(图3B)细胞的混合物低效的分离。 分离的扁桃体B和T淋巴细胞(图3A)是足够的质量为下游分析样核酸隔离。在当前的协议,DNA和…

Discussion

一个影响该协议的结果的最重要因素是使用新鲜扁桃体材料作为原料。因此,扁桃体样品应在3个小时的手术后处理。扁桃体可以从成人和儿童中获得。从孩子扁桃体材料通常是较小的由于局部手术切除扁桃体(扁桃体部分切除术)的。因此,从扁桃体部分切除术的样品(1×10 7 -1×10 8)中得到跨国公司的数目较少相比扁桃体样品(1×10 8 -2×10 9)。然而,细胞分离?…

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgströms 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.

Materials

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
100-mm petridish Nunc 172958
Dissecting foreceps Fisher Scientific 1381241
Straight iris scissors  Fisher Scientific 12912055
disposable scalpels Swann-Morton REF 0501
100μm plastic cell strainer Corning Life Sciences 352360
40μm 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 

Referências

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Assadian, F., Sandström, K., Laurell, G., Svensson, C., Akusjärvi, G., Punga, T. Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils. J. Vis. Exp. (105), e53374, doi:10.3791/53374 (2015).

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