Before the assay, cells were collected with non-enzymatic Cell Dissociation Solution (Sigma-Aldrich, Poland), centrifuged, resuspended in DMEM (with no FBS), counted in a Burker counting chamber (Roth, Germany) ABT-737 price in light microscopy with trypan, and diluted to the desired concentration. The cells were used immediately in the migration assay. Migration chamber preparation Fibronectin assay: 8-μm insert selleck chemical membranes (Falcon

BD Biosciences, USA) were sterilely covered with fibronectin (100 μg/ml, Falcon BD Biosciences). Both sides of the membrane were covered with 20 μl of the fibronectin suspension and incubated for 30 min at 37°C. Fibronectin was removed and the inserts were washed three times with sterile water. Subsequently, both sides of the membrane were immersed in a 0.1% albumin solution and incubated for 15 min. The inserts were washed three times with sterile water and dried. The prepared inserts were not stored, but used immediately

after preparation. Matrigel assay: according to the manufacturer’s instructions, the 8-μm insert membranes (Falcon BD Biosciences) were covered with matrigel diluted 1:4 with DMEM under sterile conditions, with cooling. Only the upper side of the membrane was covered with 10 μl of the matrigel suspension (i.e. approx. 7 μg/cm2 of the membrane) and slowly dried (overnight PI3K Inhibitor Library in a covered plate) at 37°C. Such prepared inserts can be stored at -20°C. If frozen, they were defrosted at 37°C, and rehydrated with DMEM for 2 hours, and directly applied in the migration TCL assay. Migration assay The cells were suspended in DMEM with no FBS, and applied to the upper section of the migration chamber, with 1 × 105 Hs294T cells/insert in both

the fibronectin and the matrigel assay, 4 × 105 B16 cells/insert in the matrigel assay, and 5 × 105 B16 cells/insert in the fibronectin assay. All preparations (bacteriophages, LPS, PBS basic control) were correlated and added at the same final volumes of PBS (125–135 μl), both in the upper and the lower sections of the migration chamber. All the preparations and cells in the upper section were completed with DMEM and with FBS-containing medium to 0.5 ml in the lower section (according to the manufacturer’s instructions). Final concentrations of the bacteriophage preparations were 1.5–2.5 × 109 pfu/ml containing 10 U/ml residual LPS. Concentration of the attracting agent, FBS, in the lower section of the migration chamber was 7.3–7.5%. The migration was carried out at 37°C with CO2. The time of migration was initially optimised and was 2 h for B16 on fibronectin, 7–8 h for B16 on matrigel, 1 h 20 min for Hs294T on fibronectin, and 4.5–5 h for Hs294T on matrigel.