Mco 19aic

The PC-3 cells obtained from ATCC (via Cedarlane, Burlington, Canada) were cultured in RPMI 1640 medium (Life Technologies Corporations) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO₂. The cells were confirmed pathogen-free via an IMPACT Rodent Pathogen Test (IDEXX BioAnalytics). Cells grown to 80–90% confluence were washed with sterile DPBS (pH 7.4) and collected after 1 min of trypsinization. The cell concentration was counted in duplicate using a hemocytometer and a manual laboratory counter.

The LNCaP cells obtained from ATCC (via Cedarlane, Burlington, ON, Canada) were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO₂. The cells were confirmed to be pathogen-free by the IMPACT Rodent Pathogen Test (IDEXX BioAnalytics). Cells were grown until 80–90% confluence and washed with sterile phosphate-buffered saline (PBS, pH 7.4) and collected after 1 min trypsinization. The cell concentration was counted in triplicate using a hemocytometer and a manual laboratory counter.

The HEK293T cells were obtained from ATCC. The FAP-expressing vector was constructed using Genome-CRISPRTM Human AAVS1 Safe Harbor Gene Knock-in Kits (GeneCopoeaiaTM) by inserting FAP-expressing gene into the AAVS1 vector. The cells were then transfected by the FAP-expressing vector following the EndofectinTM Transfection Reagent protocol. The cells underwent 3 serial dilutions and were sorted using fluorescence-activated cell sorting (FACS) to obtain FAP-expressing monoclonal colonies. HEK293T:hFAP cells were cultured in DMEM GlutaMAX™ medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO₂. Cells were grown until 80–90% confluence and washed with sterile PBS (pH 7.4) and collected.

The HEK293T:hFAP cells generated in our lab [31 (link)] were cultured in DMEM GlutaMAX™ medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO₂. Cells were grown until 80–90% confluence and washed with sterile phosphate-buffered saline (PBS, pH 7.4) and collected after 1 min trypsinization. The cell concentration was counted in triplicate using a hemocytometer and a manual laboratory counter.

For cytological studies, human cancer cell lines were chosen, with special emphasis on the colorectal, HCT116, and neuroblastoma SH-5YSY cell lines, as well as lung normal epithelial BEAS-2B and cancer H1299 and A549 cancer cell lines (all obtained from the ATTC collection; Manassas, VA, USA). The cells were harvested under the standard conditions in incubator (5% CO₂, 37 °C and 60% humidity; Panasonic model MCO-19 AIC). Cells were cultured in T75 mL sterile bottles (Sartedt), using DMEM-F12 medium (Merck, Poznań, Poland) supplemented with 10% of Fetal Bovine Serum (EURx, Gdańsk, Poland). During the experimental procedure, each cell line wasn’t more than 60–80% confluenced, no older than maximal number of passages 30. For adherent cell passages, the enzyme trypsin working solution, prepared in sodium phosphate buffer saline (PBS, pH = 7.4; PAN-Biotech Gmbh, Aidenbach, Germany), was used. The tested compounds were prepared as 1 mM stocks in 100% DMSO (Merck, Poznań, Poland), and were stored at −20 °C. Before use in cytological studies, the working solution of each compound was prepared directly in complete DMEM-F12 medium. A positive-control, anticancer drug, etoposide (Merck, Poznań, Poland), was dissolved and prepared as solutions with doses of 20, 10, 5, 2.5, 1.25, 0.63 µM (0 µM for control, untreated cells in DMEM-F12 without any tested compound additions), respectively.

The PC-3 cells obtained from ATCC (via Cedarlane, Burlington, Canada) were cultured in RPMI 1640 medium (Life Technologies Corporations) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO₂. The IMPACT Rodent Pathogen Test (IDEXX BioAnalytics) verified that the cells were pathogen-free. Cells were washed with sterile phosphate-buffered saline (PBS, pH 7.4) and collected after 1 min trypsinization when grown to 80–90% confluence. The cell concentration was counted in triplicate using a hemocytometer and a manual laboratory counter.

The LNCaP cells obtained from ATCC (via Cedarlane, Burlington, Canada) were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO₂. The cells were confirmed pathogen free by the IMPACT Rodent Pathogen Test (IDEXX BioAnalytics). Cells grown to 80-90% confluence were then washed with sterile PBS and collected after trypsinization. The cell concentration was counted in triplicate using a hemocytometer and a manual laboratory counter.