Calcein am

10 mL DMEM of media was inserted in the 3DCFS device and 6 × 10⁶ A375 cells were added. The 3DCFS was then incubated at 37 °C with 5% CO₂ on a stirrer (IKA^® Color Squid, Sigma Aldrich, Germany) at 150 rpm. Two sets of samples were taken before the cells were exposed to the 3DCFS: unstained control and stained cells after 5 min (time 0). Once in 3DCFS, the cells were sampled (300 µL) after 3, 6, 9, and 24 h, 300 µL per sample hours. All the samples were centrifuged at 1200 rpm for 5 min, washed with phosphate-buffered saline (PBS), and stained with calcein AM (Cayman Chemical, Ann Arbor, MI, USA) for 30 min at room temperature (dilute aliquot 1:1000). The results were normalized to the viability control samples taken at time 0.
For testing the recovery of cells in floating conditions, 10 mL RPMI medium was added to the 3DCFS and 7 × 10⁶ BXPC-3 cells were inserted and incubated at 150 rpm for 4 h. Then, 100 µL samples were taken after 1, 2, 3 and 4 h, seeded in 6-well plate wells with 3 mL media and incubated for 48 h. Similar protocols were used to assess cell viability post 2 mM Cisplatin treatment as detailed below.

H1299 cells growing on 96-well plates were transfected with Nsp1 mRNA as described above and incubated with the lipofectamine-RNA mix for 3 h. Little difference in gene expression was observed between 2 and 4 h of incubation with the lipofectamine-RNA mix. Media was then replaced by addition of 80 µL DMEM-10% FCS-100U/mL Pen-Strep or 80 µL serum-free DMEM-1% N2 supplement-100U/mL Pen-Strep. H1299 cells were then returned in the CO₂ incubator overnight.
Approximately 20 h after replacement of the lipofectamine-RNA mix from H1299 cells, the media was again replaced with 100 µL of a mixture containing compatible fluorophores diluted in FluoroBrite™: 1 µM Hoechst 33,342, 1 µM Calcein-AM (Cayman Chemical Company), and 20 nM Tetramethylrhodamine, ethyl ester (TMRE, Cayman Chemical Company). Cells were placed in the CO₂ incubator for 1 h and the media was changed to 50 µL of Fluorobrite or PBS alone.
Fluorescence from 96-well plates was measured using a Spectramax Microplate Gemini XPS reader with the following parameters: Hoechst 33,342 staining—excitation-355 nm; emission-460 nm; calcein-AM—excitation-485 nm; emission-520 nm; TMRE—excitation-544 nm; emission-590 nm. After normalizing values for each fluorescence reading to non-transfected controls, the product of all three readings represents the “Viability Index”.

Recombinant human TGF-β1 (referred to as TGF-β in this study) was purchased from R&D Systems (Minneapolis, MN, USA). Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) was obtained from Sigma (St. Louis, MO, USA). Calcein AM was obtained from Cayman Chemical (Ann Arbor, MI, USA). The following cell signaling inhibitors were used: SB203580 (10 μM; EMD Millipore, Burlington, MA, USA), SB431542 (10 μM; Tocris, Bristol, UK), SB505124 (500 nM, Sigma), LY294002 (2 μM, EMD Millipore), and PD98059 (10 μM, EMD Millipore). All the reagents were reconstituted according to the manufacturer's instructions.

ADCC was examined using a calcein-acetyoxymethyl (Calcein-AM; Cayman Chemical Co., Ann Arbor, MI, USA) release assay. Canine lymphokine-activated killer (LAK) cells were prepared by culturing canine peripheral blood mononuclear cells from a healthy beagle dog in the presence of 1000 IU/mL of human recombinant interleukin-2 (IL-2) (Novartis, East Hanover, NJ, USA) for 1 week, as previously reported [36 (link)]. CMM2 and KMeC cells were used as target cells. The target cells were labeled with Calcein-AM for 30 min, washed 3 times with PBS containing 5% FBS, and plated onto 96-well plates at a density of 1 × 10⁴ cells/well. P38Bf or whole molecule dog IgG was added at various concentrations from 0.01 to 10 μg/mL for 15 min on ice. Next, the LAK cells were added as effector cells at an effector (E)/target (T) ratio of 10:1. Then, the plates were incubated for 4 h at 37 °C, and the relative light units (RLU) of the supernatants were analyzed using fluorometry to measure calcein release (cell death). For maximal release, the cells were lysed with 2% Triton X-100. Fluorescence was detected using the ARVO X4 system (PerkinElmer, Waltham, MA, USA). ADCC activity was calculated using the following formula:
For each experiment, measurements were conducted in quadruplicate using four replicate wells. Each experiment was repeated at least 3 times.

For the staining, 0.2 μM Calcein AM (Cayman Chemicals, Ann Arbor, MI, USA) was added to the cells in the medium. After an incubation for 15 min at 37 °C, the staining solution was removed, and the cells were washed twice with PBS. The neurospheres were visualized under the microscope (ZOE Fluorescent Cell Imager, Bio-Rad Laboratories GmbH, Feldkirchen, Germany). Fluorescence intensity was measured in lysates (Passive lysis buffer, Promega, Walldorf, Germany) of the neurospheres at 485 ex/520 em nm. For size determination, the diameter of six neurospheres per animal (measured horizontal) was determined using Fiji/ImageJ software (NIH, Bethesda, Rockville, MD, USA).

The viability of SkM on HAM and EF-HAM scaffolds was assessed through Live/Dead assay which distinguishes live from dead cells through staining with Calcein AM (Cayman Chemical, Ann Arbor, MI, USA) and ethidium homodimer-1 (EthD-1; Thermo Fisher), respectively. SkM were seeded on scaffolds at a 10,000 cells/cm² seeding density and cultured for 48 h before staining procedure. For staining, cells were incubated with a mix solution of 2 µM of Calcein AM and 4 µM of EthD-1 prepared in Hank’s 1X Balanced Salt Solutions (HBSS; Cytiva, Marlborough, MA, USA) at room temperature for 30 min. Staining solution was then replaced with HBSS and the images of stained cells were captured at five different fields using fluorescence microscope (Nikon, Tokyo, Japan) before the number of live and dead cells were counted and averaged.