Casy cell counter

The left lung lobe was cut into small pieces and incubated in digestion buffer for 45 min at 37 °C in a shaking water bath. The digestion buffer was RPMI 1640 medium (Lonza, Verviers, Belgium) containing 10% fetal calf serum (Lonza), 0.7 mg/ml collagenase A (Sigma-Aldrich, Zwijndrecht, The Netherlands) and 10 μg/ml DNAse I (grade II from bovine pancreas, Roche Applied Science, Almere, Netherlands). After digestion, the lung tissue was forced through a 70 μm nylon strainer (BD Biosciences, Breda, Netherlands) to obtain single lung cell suspensions. A 2-minute incubation with 10 times diluted Red Blood Cell lysis buffer (Biolegend, Fell, Germany) was done to lyze erythrocytes followed by centrifugation through 70 μm strainer caps. Cells were counted using a Casy cell counter (Roche Innovatis AG) and were ready for flow cytometric stainings.

For I-SceI spike-in we used a yeast strain (I-SceI strain) with GAL-inducible I-SceI endonuclease and a single I-SceI-cutting site integrated at the ADH4 locus on chromosome VII. To measure the cleavage efficiency of I-SceI, cell aliquots were taken before (RAFF) and 2 h after (GAL) cleavage induction, and total gDNA was extracted. DNA was serially diluted and amplified for 25 cycles with primers spanning the I-SceI cutting site. Cleavage efficiency was inferred by comparing the amount of amplified DNA in GAL (cut) vs. RAFF (uncut) conditions. We used CASY Cell Counter (Roche Applied Science) to mix this spike-in with our sample of interest (wild-type cells with replication stress induced by HU treatment) in proportion 2:98. The cutting ratio of the I-SceI endonuclease expressed in the I-SceI strain was estimated using an unmixed I-SceI strain and Equation (2) below.

Heparinized peripheral blood was obtained from healthy donors, after giving informed consent, and diluted in a 1:1 ratio with phosphate-buffered saline (PBS) containing 10% (v/v) trisodium citrate (TSC). Immune cells were separated from each other by density gradient centrifugation over isotonic Percoll (Pharmacia, Uppsala, Sweden, 1.076 g/mL) at 800g at room temperature (RT). The interphase fraction, containing peripheral blood mononuclear cells (PBMCs), was collected for isolating T cells and/or monocytes. Cells were purified using magnetic-activated cell sorting (MACS) with either the Pan T cell isolation kit or the Pan monocyte isolation kit from Miltenyi-Biotec (Bergisch Gladbach, Germany) and used according to the manufacturer’s instructions. Polymorphonuclear cells (PMNs) were isolated from the pellet fraction after consecutive erythrocyte lysis by adding lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 Mm EDTA) at 4°C. Subsequently, cells were resuspended in HEPES+ medium (containing 132 mM of NaCl, 20 mM HEPES, 6.0 mM KCl, 1.0 mM MgSO₄, 1.0 mM CaCl₂, 1.2 mM potassium phosphate, 5.5 mM glucose, and 0.5% (w/v) human serum albumin, pH 7.4). Cell concentrations were determined by the CASY Cell Counter (Roche, Basel, Switzerland).

Twenty-three healthy volunteers were recruited at the University of Manchester as part of the National Repository Healthy Volunteers (NRHV) study. All the samples were collected with ethical committee approval (MREC 99/8/84) and all individuals provided informed consent. The median age of the subjects was 50.5 years (range=26–82 years) with eight males and 15 females. A total of 20 ml peripheral blood was collected in Vacutainer plus tubes containing EDTA (Becton Dickinson). PBMCs were extracted within 2 h of collection from 20 ml of whole blood using Ficoll Plus density gradient centrifugation (GE Healthcare). Cells were washed twice with MACS running buffer (phosphate-buffered saline (PBS), bovine serum albumin, EDTA and sodium azide; Miltenyi) and total PBMCs counted using the CASY cell counter (Roche). To allow collection of all the samples, PBMC samples were immediately cryopreserved in 1 ml recovery cell-freezing medium (Gibco) per 5 × 10⁶ cells at a cooling rate of −1 °C per minute. Samples were genotyped using the Illumina HumanCoreExome v1.0 array, in accordance with the manufacturer’s instructions. Genotype clustering and calling was performed using the GenomeStudio Data Analysis software platform using the Illumina HumanCoreExome-24 v1.0 Manifest File.

IFN-γ ELISpot assays were performed according to the manufacturer’s protocol (U-Cytech). Spleens were homogenized and passed through 70 μm filters (BD Biosciences), washed with RPMI 1640 containing 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin and 292 μg/ml glutamine and counted using a Casy cell counter (Roche). Cells were plated in a concentration of 4*10⁵ cells/well in an IFN-γ antibody-coated PVDF membrane plate (Millipore MSIP) and stimulated with 0.1 nmol/well of either WT peptide or corresponding CPL. After 16 hours of incubation spots were visualized according to the manufacturer’s protocol (U-Cytech) and counted using an A.el.vis reader (Sanquin).