Macs tumor dissociation kit

Surgical specimens were obtained by the core needle biopsy of surgically removed tumors. The specimens were dissociated into single cells using a MACS Tumor Dissociation Kit and a gentle MACS dissociator (Miltenyi Biotec, North Rhine-Westphalia, Germany) in accordance with the manufacturer’s instruction. For pleural effusion specimens, drainage fluid was collected after trocar placement, centrifuged, and purified with Red Blood Cell Lysis Solution lysis buffer (Miltenyl Biotec). The organoids were established from the obtained cells as described previously [8 (link)]. Briefly, cell pellets were resuspended in BME gel (R&D Systems, Minnesota, USA) at 4 °C and seeded to form domes in 24-well or 48-well plates (IWAKI, Tokyo, Japan), polymerized at 37 °C for 10–20 min, and the prepared medium was added at a volume of 500 mL per 50 µL BME dome. The medium was changed every 3–4 days and the PDO was passaged depending on growth. Cultrex Organoid Harvesting Solution (Thermo Fisher Scientific, Massachusetts, USA) and 0.25 w/v% Trypsin-1 mM EDTA 4Na Solution with Phenol Red (FUJIFILM, Tokyo, Japan) were used for passaging of organoids. Expression of ER and PgR in surgical specimens was assessed using the Allred score [12 (link)].

For apoptotic cell determination, the tumors were dissociated to single cells using the MACS Tumor Dissociation Kit in combination with the gentleMACS Dissociator (Miltenyi Biotec) as according to the manufacturer’s protocol. After dissociation, the cells were filtered through a 30 µm MACS SmartStrainer. Cells were pelleted from the filtrate at 300 g x 7 min and resuspended in 400 µl Binding Buffer. The cells were incubated with Annexin V FITC and Propidium Iodide (Sigma Aldrich) for 15 min in the dark at room temperature. After incubation, the cells were pelleted and resuspended in 100 µl Binding Buffer for analysis by flow cytometry using BD Accuri C6 cytometer (BD Biosciences, CA, USA).