Iso osmotic sodium citrate solution

Pancreatic cancer cell line HPAF-II was obtained from American Type Culture Collection (ATCC, Virginia, USA), and grown using EMEM medium (ATCC) supplemented with 10% fetal bovine serum (ATCC) and 100 U/mL penicillin-streptomycin (ATCC). All cell lines were grown at 37 °C with 5% CO₂ and were routinely passaged at 80% confluence using an iso-osmotic sodium citrate solution for cell release (Thermo, Massachusetts, USA).

In preparation for laser microdissection, cells were released from the culture plates using the iso-osmotic sodium citrate solution (Thermo). Following a wash with the culture medium, each cell line was diluted to a density of 1000 cells per 100 μL. Approximately 1000 cells (100 μL) were smeared on PEN membrane slides (Leica, Wetzlar, Germany), air-dried for 10 min, and fixed with 100 μL of 100% ethanol. Cells were then isolated using the PALM MicroBeam laser microdissection system (Zeiss, Oberkochen, Germany). 1, 5, 10 or 20 cells were laser microdissected and collected into 200 μL opaque tube caps (Zeiss) using the laser pressure catapult function. Cell transfer to the tube cap was confirmed by imaging the cap prior to cap closure using the cap-check function.