Saponin

Intracellular staining was performed after incubation with brefeldin A (1 μg/ml, Sigma-Aldrich, Schnelldorf, Germany) for 4 h. Subsequently, the cells were surface stained and fixed of the cells with PBS/ 4% paraformaldehyde. Then cells were permeabilized with 0.5% saponin (Carl Roth, Karlsruhe, Germany) and intracellularly stained with IFN-γ-Alexa647 (BD Bioscience, Pharmingen, Heidelberg, Germany). To determine the expression of the activation marker CD69, the cells were harvested and stained in PBS containing 2% FCS (Thermo Fisher Scientific, Oberhausen, Germany) with CD69-PE-Cy7, CD3-Horizon V450, CD56-PE (BD Pharmingen, Heidelberg, Germany) for 15 min at 4°C in the dark, washed and analyzed via flow cytometry. Degranulation of NK cells was determined by measuring CD107a-PE, CD3-FITC, CD56-APC (BD Pharmingen, Heidelberg, Germany) and Vγ9-Alexa488 (for γδT cell staining (78 (link)) as generous gift from Dietrich Kabelitz and Daniela Wesch). Human PBMCs were co-cultured with tumor cells and CD107a-PE was added for 1 h. Degranulation was blocked with 5 μg/ml Monensin A (Sigma-Aldrich, Schnelldorf, Germany) for an additional 3 h. Flow cytometric analysis was performed using the LSR II from BD Bioscience and data were analyzed with FlowJo® (Tree Star, Switzerland).

Immunocytochemistry was carried out on paraformaldehyde-fixed cells grown on glass cover slips as described earlier [4] (link). In brief, cells were permeabilized and blocked with 3% BSA and 0.1% saponin (both Roth, Karlsruhe, Germany) in PBS, stained with the primary antibodies diluted in blocking buffer for 1 h at room temperature or overnight at 4°C following incubation with the secondary antibody diluted in blocking buffer for 30 min at room temperature. Between all steps cells were triple washed with 0.1% saponin in PBS and then mounted on glass microscopic slides in ProLong Gold anti-fade reagent (Invitrogen Life Technology). The images were taken with an Axioplan II fluorescence microscope and AxioCam MRm camera and processed by Axiovision 4.7 (all Zeiss, Oberkochen, Germany). All samples were counted blind.

Surfactant‐like liposomes were prepared and transport of TopFluor‐labeled phosphatidylcholine (TopF‐PC) into HA‐positive vesicles was quantified as described before.12 In short, TopF‐PC containing liposomes (1:20 diluted in OptiMEM, Thermo Fisher Scientific) were offered to the cells expressing WT or mutant ABCA3‐HA for 30 minutes at 4°C. After two hours at 37°C, cells were treated with potentiators or DMSO as a vehicle control for 24 hours. Then cells were covered with 5% BSA (in PBS) for 30 minutes at 4°C for removal of residual labelled lipids adherent to the cell membrane. Cells were fixed, permeabilized with saponin (Carl Roth GmBH, Karlsruhe, Germany) and stained for HA‐tag. Microscopy, fluorescence analysis and quantification of vesicle volume and percentage of filled vesicles were performed as described previously12 using a confocal laser‐scanning microscope (LSM 800, ZEISS with ZEN 2 blue edition software) and the modified Fiji (Image J) plugin “Particle_in_Cell‐3D”.35

In order to detect IFNγ, TNFα, and Granzyme B produced by CD8⁺ T cells in tumor-infiltrating leukocytes (TILs), enriched TILs from MC38 tumors were incubated for 6 h at 37 °C with 0.5 mg/ml PMA (Sigma-Aldrich, St. Louis, MO) plus 0.05 mg/ml ionomycin (Sigma-Aldrich, St. Louis, MO) or with medium alone, in the presence of 5 mg/ml Brefeldin A (Sigma-Aldrich). Afterwards, cell surface markers were stained as described above and cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% saponin (ROTH, Karlsruhe, Germany) in FACS buffer and stained intracellularly for the respective cytokines. Samples were analyzed on either a LSR-II, FACSCanto II, or a FACSSymphony (Becton Dickinson, Mountain View, CA), and data were analyzed with FlowJo Analysis Software (Tree Star Inc, Ashland, OR).