Luna cell counter

For analysis of cell death, cells were seeded in T25 flasks. Cell numbers were adjusted for each cell line to account for varying proliferation rates (1 × 10⁵ CAL72 cells, 1.4 × 10⁵ SAOS2 cells, and 0.8 × 10⁵ cells for HeLa, HCT116, and U2OS). The following day each cell line was either treated with 3 μM VE-821 (Selleckchem) or with the same volume of DMSO for the control samples. Cells were incubated for 6 days without medium change. Cells, including dead cells, were collected by trypsin and total cell numbers were determined using the LUNA cell counter (Biozym). Cells were resuspended in FACS binding buffer (10 mM HEPES, 2.5 mM CaCl₂, 140 mM NaCl) at a final concentration of 2 × 10⁶ cells/ml, stained with FITC annexin V (BioLegend) and propidium iodide (Miltenyi Biotec) according to the manufacturers’ instructions, and analyzed by flow cytometry on a FACS Canto II (BD Biosciences). The fraction of apoptotic cells, characterized as annexin V positive, was quantified using the FACS Diva software. The percentage of induced cell death d_ind was calculated as
$d_{\text{ind}}=\frac{\left[\left(\begin{array}{l}\text{percentage dead }\\ \text{cells treated}\end{array}\right)-\left(\begin{array}{l}\text{percentage dead }\\ \text{cells control}\end{array}\right)\right]}{\text{percentage viable cells control}}\times 100\%.$

Permanent cell lines BxPC3, MIA PaCa-2 and AsPC-1 were obtained from the American Type Culture Collection (Rockville, MD, USA). The BH1362 KRAS G12C NSCLC cell line was established from a pleural effusion according to the Ethics Committee EK-21-210-1221 of the Viennese Hospital Association. Aforesaid cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS (Biochrome, Berlin, Germany) and Penicillin/Streptomycin (Sigma-Aldrich). Upon confluence cells were detached with trypsin/EDTA (Sigma-Aldrich) and counted with a LUNA cell counter (Biozym, Vienna, Austria).

The breast cancer cell lines MDA-MB-231, MDA-MB-436, T47D and HCC1937 were obtained from the ATCC (Rockville, MD, USA), cultured in RPMI-1640 medium and upon confluence, cells were detached with trypsin/EDTA (Sigma-Aldrich) and cells counted with a LUNA cell counter (Biozym, Vienna, Austria). MDA-MB-231, MDA-MB-436 and T47D have been established from pleural effusions and and HCC1937 from a local tumor.