Fluorescence mounting solution

Purified monocytes and NK cells (5 × 10⁴ cells per coverslip) were seeded on poly‐L‐lysine coated coverslips. After 4 h at 37°C, the cells were washed twice with PBS and then fixed with 4% paraformaldehyde for 15 min at room temperature. Coverslips were washed six times with cold PBS and incubated with blocking/permeabilization solution (10% mouse serum, 0.2% Triton X‐100 [Sigma] in PBS) for 1 h at room temperature. After this step, cells were incubated overnight with the anti‐P2X7 rabbit polyclonal primary antibody (1:100 dilution) at 4°C. Then, cells were washed with PBS and incubated with AlexaFluor 488 donkey anti‐rabbit IgG secondary antibody (1:800 dilution) for 1 h at room temperature, rinsed in PBS and finally incubated for 10 min with DAPI (1 μg·ml⁻¹). All coverslips were mounted on slides with Fluorescence mounting solution (DAKO). Images were acquired using a Nikon Eclipse Ti microscope equipped with ×40 S Plan Fluor objective (numerical aperture, 0.6) and 60×/0.60 S Plan Fluor objective and a digital Sight DS‐QiMc camera (Nikon, Tokio, Japan) with a Z optical spacing of 0.4 μm and 387‐/447‐nm, 472‐/520‐nm filter sets (Semrock). Maximum‐intensity projection of images was achieved with NIS‐Elements AR software (Nikon) and ImageJ software (US National Institutes of Health).