Nylon mesh cell strainer

Experiments were performed on larvae 1–2 days post hatching and thus a mix of first and early 2^nd instar larvae. The water in which larvae were swimming was passed through a 100 μm Nylon mesh cell-strainer (Fisher Scientific) to concentrate the larvae. This concentrated suspension of larvae was diluted until 100 μL of suspension contained 5–10 larvae. A 100 μL aliquot of this suspension was added using a standard Gilson pipette to each well of a 96-well plate, with the tip of the pipette being cut back to reduce damage to the larvae. Subsequently, 100 μL of the compound under test was added (dissolved in water from a 10^-2M DMSO stock to yield the required final concentration of 10^-4M). Wells containing DMSO alone diluted to appropriate concentrations served as controls.

Ventricular cardiac myocytes were isolated from male WT and K229Q mice. Mice were injected with heparin (200 UI/kg) to prevent blood coagulation, deeply anesthetized with 5% isoflurane (confirmed by abolished pain reflexes), and subjected to cervical dislocation prior to thoracotomy. Hearts were quickly excised via aorta, pulmonary and vena cava transection, and cannulated (21 gauge) onto a gravity fed, constant-pressure Langendorff perfusion system (Radnoti). All perfusates were kept at 37 °C. Each heart was perfused with Ca²⁺-free Tyrode’s solution (mM: 136 NaCl, 5.4 KCl, 10 HEPES, 1.0 MgCl₂, 0.33 NaH₂PO₄, 10 Glucose, pH 7.4) for ~5 min, followed by perfusion of Ca²⁺-free Tyrode’s enzyme solution containing collagenase (Type 2, 1 mg/ml, Worthington) and protease (Type XIV, 0.1 mg/ml, Sigma) for 10–13 min. The enzyme solution was washed out for ~8 min with Tyrode’s solution with 0.1 mM CaCl₂. After excision, ventricular tissue was placed in a petri dish containing 0.1 mM CaCl₂ Tyrode’s solution and then gently shredded with forceps. Cardiac ventricular myocytes were separated from dead and contaminant cells via filtration through a 100 μm sterile, nylon mesh cell strainer (Fisher). Single cells were then reintroduced to 1.0 mM CaCl₂ for 15 min and then stored for up to 5 h in 1.8 mM CaCl₂ at room temperature.

Tumor suspensions were obtained after mechanical and enzymatic disaggregation (Accumax (Merck Millipore, Burlington, MA, USA) (15 min, room temperature (RT)) and filtered through 70 μM nylon mesh cell strainer (Thermo Fisher Scientific). Erythrocytes were lysed with Quicklysis buffer (Cytognos, Salamanca, Spain) and cells were incubated with hFcR Blocking (Miltenyi Biotec, Bergisch Gladbach, Germany), previous to antibody (Table S2) incubation (20 min at 4 °C in PBS 1% fetal bovine serum (FBS)). Cells were labelled with a Fixable Viability Stain (Becton Dickinson, Franklin Lakes, NJ, USA) (20 min, RT). The analysis was conducted in a Macsquant10 flow Cytometry (Miltenyi Biotec). Subset definition was: Neutrophils: CD45⁺CD11b⁺CD16⁺CD15⁺CD14⁻CD33⁻; myeloid-derived suppressor cells (MDSCs): CD45⁺CD11b⁺CD16⁺CD15⁻CD14^+/−CD33⁺; Macrophages: CD45⁺CD11b⁺CD16⁻CD15⁻CD14^+/−CD33⁻MHCII⁺; Tregs: CD45⁺CD3⁺CD4⁺CD25⁺CD127^lo.

EGFP-positive pectoral fin cells were isolated from zebrafish embryos using FACS. Tg(Mmu:Prx1-EGFP) embryos were collected at 24 hpf (CUT&RUN) and 48 hpf (RNA-seq) and digested in Pronase (1 mg/ml) (Roche) for 5-6 min to remove the chorion. Embryos were pooled together, washed in DPBS (Gibco), and dissociated in Accumax (Innovative Cell Technologies) and DNase I (50 U/100 embryos) (Roche) at 31°C for 1.5 h. Cells were homogenized every 8 min by pipetting up and down using pipette tips decreasing in size. Cells were washed in solution (300 U DNase I in 4 ml DPBS) before filtering through a 70 μm nylon mesh cell strainer (Thermo Fisher Scientific) into 50 ml conical tubes pre-coated with 5% fetal bovine serum (FBS; Invitrogen). Cells were spun down, resuspended in basic sorting buffer [1 mM EDTA, 25 mM HEPES (pH 7.0), 1% FBS in DPBS], stained with DAPI (1:1000), and sorted using FACS at the University of Colorado Cancer Center Flow Cytometry Shared Resource (Aurora, CO, USA) on the MoFlo XDP100 sorter (Beckman Coulter) with a 100 μm nozzle tip (Beckman Coulter).

Following vaccination, immune cells were isolated from the spleens, lungs, PP of the gut and FRT.
Immune cells from the spleen and PP were isolated by pressing harvested tissue through a 70-µm sterile nylon-mesh cell strainer (Thermo Fisher Scientific) using a 5-ml syringe rubber plunger. Following the removal of red blood cells with ACK lysis buffer (0.14 M NH₄Cl, 10 mM KHCO₃ and 100 mM Na₂EDTA), cells were washed and resuspended in R10 (RPMI 1640 supplemented with 1% P/S, 10% FBS, and β-mercaptoethanol). Cells were counted using a CASY cell counter (Termo Fisher Scientific).
To obtain immune cells from the lungs and female genital tract, the dissected organs were cut into 1-mm² segments and digested with 1.4 mg/ml Collagenase (Sigma Aldrich, Gillingham, UK) and 60 µg/ml DNase Type IV (Sigma Aldrich) in R0 supplemented with β-mercaptoethanol in 1.8-ml volume for 60 min. at 37 °C with shaking, after which 200 µl of FBS was added to quench the reaction. The cells were then processed in the same manner as those isolated from the spleen and Peyer’s patches.