Cell stripper

BMDMs were loaded with FLICA 660 Caspase-1 Assay (final dilution of the FLICA 660 reagent for incubation with cells was 1:300, ImmunoChemistry) after 1h of treatment with nigericin and incubated for additional 1h. Cells then were washed twice with washing buffer and harvested using Cell Stripper (GIBCO). Collected cells were centrifuged and resuspended in annexin binding buffer (10 mM HEPES, 0.14 M NaCl, 2.5 mM CaCl₂, pH 7.4) containing AnnexinV-FITC (Biolegend) and LIFE/DEAD Aqua dead cell stain (Thermo Fisher) and incubated for 15 min. Cells were diluted 1:3 with annexin binding buffer and acquired with a BD CantoII. Results were acquired with the FACSDiva software (BD) and FLICA⁺AnnexinV⁺ population was quantified using FlowJo software (Tree Star).

Cells were seeded into monolayer or suspension for 7 days, at which point the cells were isolated, using a non-enzymatic dissociation buffer (CellStripper, Invitrogen, Carlsbad, CA, USA), and re-seeded into either the tissue culture polystyrene or the Ultra-Low Attachment 96 well plates, at a concentration of 10,000 cells/well. Cells were maintained in a complete medium, overnight, prior to the TRAIL treatment. The cells were treated with the recombinant human TRAIL (TRAIL) (R&D Systems, 375-TEC), at two different doses (10 ng/mL and 100 ng/mL for MDA-MB-231 and ZR75-1 cell lines; 50 ng/mL and 500 ng/mL for MCF7 cell lines). The concentrations were based on our previous research, in which the IC50 for TRAIL treatment over 24 h was found to be 1–5 ng/mL for MDA-MB-231, 7–8 ng/mL for ZR75-1, and >500 ng/mL for MCF7 cells [37 (link)]. Plates were incubated with TRAIL over 24 h and analyzed for either viability or protein expression, at indicated timepoints.

The B16F10 mouse melanoma line was originally obtained from I. Fidler (MD Anderson Cancer Center, Houston, TX). The 4T1 mouse breast cancer cell line and CT26 colon carcinoma were purchased from ATCC (Manassas, VA). WG492 is a melanoma cell line derived from a tumor from the BRAF^V600E/PTEN^−/− transgenic mouse. These cells were maintained in RPMI 1640 containing 7.5% fetal bovine serum (FBS) and l-glutamine. Cells were detached using 0.25% trypsin/EDTA. For cell surface staining of tumor cells and CD8⁺ T cell killing assays, cells were detached non-enzymatically using Cellstripper (Invitrogen).

SKBR3 cells were obtained from ATCC. Biotin and fluorescein conjugated Anti-HER2 Affibody Molecule (HER2-AFF-B, termed: (ZHER2:477)2) was purchased from Affibody AB, Bromma, Sweden. Dulbecco’s Phosphate Buffered Saline (DPBS), Dulbecco’s Modified Eagle’s Medium GlutaMAX^TM with high glucose and pyruvate (DMEM), Fetal Bovine Serum, certified, heat inactivated (FBS), Minimum Essential Medium Non-Essential Amino Acids (NEAA) 100× solution, Phosphate Buffered Saline (PBS) 10× solution, CellStripper, Normal Goat Serum (GS), and Quantum Dot (QD) Qdot 655 Streptavidin Conjugate (Strep-QD) were from Fisher Scientific GmbH, Schwerte, Germany. D-Saccharose, Glycine, biotin free and molecular biology grade Albumin Fraktion V (BSA), and Sodium Cacodylate Trihydrate were from Carl Roth GmbH + Co. KG, Karlsruhe, Germany. Electron microscopy grade Formaldehyde (FA) 16% solution was from Science Services GmbH, Munich, Germany. HPLC grade deionized Water, 0.01% poly-L-lysine (PLL) solution (mol wt 70,000–150,000), Sodium Tetraborate, Boric Acid, and Fibronectin-like Engineered Protein Polymer-plus (FLEP), were from Sigma-Aldrich Chemie Gmbh, Munich, Germany. 35 mm-cell culture dishes with uncoated glass bottom were from Greiner bio-one, Frickenhausen, Germany (CELLviewTM four compartments), and from ibidi, Munich, Germany (μ-Dish, with imprinted 50 or 500 μm relocation grid).