70 μm mesh

The clinical material comprised tonsillar tissue from 78 subjects: 31 children and 47 adults (Fig. 1). The pediatric donors ranged in age from 2 to 15 years (average, 6.6), and the adults from 16 to 69 (average, 30.3). Of the specimens, 31 were from males and 47 from females (Fig. 1). The tonsillectomies and tonsillotomies were performed in most cases due to chronic tonsillitis or tonsillar hypertrophy (Fig. 1). All tissues were collected and used in accordance with the ethical rules of the Ethics Committee of the Hospital District of Helsinki and Uusimaa. All tonsil tissues were freshly obtained directly after surgical resection at the operation theatre. The tonsils were cut with disposable scalpels and cell suspensions were prepared by mechanical homogenisation with a syringe plunge, followed by a wash with PBS and filtration through a 70 μm mesh (Corning Life Sciences). The cells were resuspended into final volume of 100 μL PBS.Fig. 1

Tonsillar samples from children and adults; clinical indications

Cells were seeded in six-well plates on the day before treatment and BET inhibitor or DMSO vehicle was added for the indicated time and dose. Cells were subjected to 10 μM EdU 6 h before staining. Viable attached cells were then washed once with cold phosphate-buffered saline (PBS) and detached using Trypsin/EDTA solution (Biochrom), resuspended and filtered through a 70-μm mesh (Corning, New York, NY, USA). The EdU⁺ population was determined using Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Life Technologies) and stained with DAPI at 1 μg/ml final concentration for 1 h at 4 °C in the dark before flow cytometer analysis.

Monolayer cells and MTS after six days of culture were dispersed using Accumax (#SCR006 Chemicon) in EMEM medium with 5% fetal bovine serum and filtered through a 70 μm mesh (# 352350, Falcon Corning, Tewksbury, MA, USA). The cell cycle was determined by flow cytometry using the DNA Reagent Kit (Cycletest Plus #340242, BD, Billerica, MA) following the manufacturer’s recommendations. The samples were processed in a BD FACSAria Cell Sorter 3 and analyzed using the ModFit LT 3.2 version software. Each individual assay was performed in triplicate. Statistical comparisons were performed using t-tests.