Isolation of Mononuclear Cells from Tonsillar Tissue

Abstract

Located on the inside of the throat, the paired palatine tonsils form part of the first major barrier protecting the digestive and respiratory tracts from potentially invading microorganisms. The tonsils have a surface of stratified squamous epithelium that extends into deep and branched crypts lined by reticulated epithelium, which in parts may only be one cell thick. Organized in the sub-epithelial space are B cell rich lymphoid follicles. T cells are mostly located in the extra-follicular spaces with a very high CD4:CD8 T cell ratio. In addition to the T and B cell subsets, six phenotypes of dendritic cells (DC) have been identified in the tonsils: Langerhans cells in the squamous epithelium, germinal center DC, and follicular DC in the germinal center, and another three DC phenotypes that are located in the extra-follicular area (interdigitating DC, plasmacytoid DC, and lympho-epithelial symbiosis-DC). Here, we describe the isolation of tonsil mononuclear cells from fresh human tonsil.

Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

“…CD4+ T lymphocytes were then isolated by positive selection using anti-CD4 microbeads (Miltenyi Biotec) [2]. CD4+ T lymphocytes were also purified from tonsil tissues [124]. The purity of CD4+ T lymphocytes—~98%—was verified by flow cytometry.…”

A Novel Bromodomain Inhibitor Reverses HIV-1 Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association

“…Jurkat cells were transduced with VSV-G pseudo-typed HIV-1 NL4-3-Luciferase virus for 8 h and treated with compounds (UMB-32, UMB-136, JQ1, or prostratin) for 24 h. Cells were collected and subjected to mRNA extraction (RNeasy® Mini Kit, Qiagen) and reverse transcription (iScript™ cDNA Synthesis Kit, Bio-Rad). The qPCR assays were conducted as previously described with slight modification (Johnston et al, 2009), using the iTaq™ Universal SYBR® Green Supermix (Bio-Rad) on the CFX Connect™ Real-Time PCR System (Bio-Rad). The following qPCR primers were used: Initiation (10–59 bp) (forward, 5′- GTT AGA CCA GAT CTG AGC CT-3′; reverse, 5′-GTG GGT TCC CTA GTT AGC CA-3′); Elongation (29–180 bp) (forward, 5′-TGG GAG CTC TCT GGC TAACT-3′; reverse, 5′-TGC TAG AGA TTT TCC ACA CTG A-3′).…”

“…Tonsils were isolated from HIV-negative donors, with obstructive sleep apnea (OSA), whom required surgical removal of their tonsils. Isolation of tonsillar mononuclear cells (TMC) was performed as previously described (Johnston et al, 2009). In brief, fresh, healthy tonsils kept in ice-cold 1x HBSS with antibiotics (100 U/mL penicillin, 100 ug/mL streptomycin, 5 ug/mL gentamicin, 0.5 ug/mL amphotericin) were cut into 3- to 10-mm fragments with scalpels then pushed through a 3-inch stainless steel sieve using the flat end of a 5-mL plastic syringe plunger.…”

Improvement of Psoriasis after Tonsillectomy Is Associated with a Decrease in the Frequency of Circulating T Cells That Recognize Streptococcal Determinants and Homologous Skin Determinants

“…Briefly, PBMCs or tonsillar mononuclear cells were isolated from heparinized venous blood of psoriatic individuals or tonsillar tissue (24) by Ficoll (Sigma-Aldrich, St. Louis, MO) density gradient sedimentation. Singlecell suspensions of tonsillar mononuclear cells were prepared as described (24).…”

“…Briefly, PBMCs or tonsillar mononuclear cells were isolated from heparinized venous blood of psoriatic individuals or tonsillar tissue (24) by Ficoll (Sigma-Aldrich, St. Louis, MO) density gradient sedimentation. Singlecell suspensions of tonsillar mononuclear cells were prepared as described (24). The PBMCs or tonsillar mononuclear cells were cultured at a density of 1 3 10 6 cells/ml in complete RPMI 1640 in cell culture tubes (Nunc, Thermo Fisher Scientific, Roskilde, Denmark) and stimulated for 16 h with the 16 peptide pools (see Supplemental Table I) at a final concentration of 2 mg/ml, in the presence of the costimulatory Abs to CD28 and CD49d (1 mg/ml each; Serotec Scandinavia, Oslo, Norway).…”