Non-adherent pre-B Nalm6 cells, REH cells and mononuclear cells from human bone marrow were collected by centrifugation. The cells were washed in cold 1X Phosphate Buffer Saline (PBS) (#ET330, Euromedex) and diluted to reach the concentration of 320,000 cells/mL. The Superfrost™ Menzel-Glaser microscope slides (#10143560 W90, Thermo Fisher Scientific) and disposable sample chambers (Shandon™ Cytofunnel™ double, 28mm2, #5991039, Thermo Fisher) were placed into appropriate slots in the Shandon Cytospin® 4 Cytocentrifuge (Thermo Scientific). Then, 250 μL of cell suspension were aliquoted in each chamber to drop approximately 80,000 cells per spot. The optimal cellular confluence for PLA experiments is 40–70% confluency after cytospin. The cytocentrifugation was run at 800 rpm during 5 min under low-acceleration. Then, chambers were removed and the areas with cells were encircled using a hydrophobic delimiting pen (Dako pen, #S200230–2, Agilent). The samples were fixed with 20 μL of 4% paraformaldehyde for 20 min at 4 °C and were washed twice in 1X PBS for 5 min in a staining jar (spots must be well covered) under shaking (60–90 rpm/min). We typically used a 70 mL staining jar for 5 slides. After fixation, the samples should not be left to dry at any case before the final step. The cells were blocked and permeabilized by adding 5% Bovine Serum Albumin Fraction V (BSA, #10735094001, Roche) and 0.2% saponin (#47036, Sigma Aldrich) in 1X PBS for 1 h 20 at 4 °C in a humidity chamber. A washing step was finally performed before incubation with primary antibodies in PBS/0.2% saponin for 5 min under shaking at room temperature in a staining jar.