Calcein am solution

BEAS-2B cells were treated with licochalcone A and stimulated with TNF-α/IL-4 for 24 h. THP-1 human monocytic cells were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan) and cultured in RPMI 1640 medium supplemented with 10% FBS and 100 U/mL penicillin and streptomycin. THP-1 cells were stained with calcein-AM solution (Sigma) for 30 min. BEAS-2B cells were co-cultured with THP-1 cells, and adherent THP-1 cells were observed by fluorescence microscopy (Olympus, Tokyo, Japan) as described previously [30 (link)].

Calcein Assay was used to determine cells viability after infection and treatments and to normalize measurements from ELISA assays. 200ul of each sample was resuspended in medium without phenol red and dispensed in black 96-culture plate in triplicate. After washing, cells were incubated for 1 hour at 37° in 4 μM Calcein-AM solution (Sigma-Aldrich, St. Louis, MO, USA). Finally, fluorescence was measured at an excitation wavelength of 494 nm and an emission wavelength of 530 nm, in a plate reader. The percentage of viable cells was calculated using the following formula:% Live Cells = [F(530)_sam–F(530)_min] × 100%, F(530)_max.
where F(530)_sam is the fluorescence at 530 nm in the experimental cell sample labeled with Calcein AM; F(530)_min is the fluorescence at 530 nm in a sample not labeled with Calcein AM (cell auto-fluorescence) and F(530)_max is the fluorescence at 530 nm in a sample where all or nearly all cells are alive, labeled with Calcein AM (sample control).

Human bronchial epithelial BEAS-2B cells were cultured in DMEM/F12 and seeded into 6-well plates. BEAS-2B cells were pre-treated with casticin (5–20 μM) for 1 h, and then incubated with 10 ng/ml TNF-α for 24 h as described previously (Huang et al., 2016 (link)). Human monocytic THP-1 cells (5 × 10⁶/ml) (Bioresource Collection and Research Center, Taiwan) were incubated with calcein-AM solution (Sigma) for 0.5 h. Next, THP-1 cells were co-cultured with BEAS-2B cells and observed under a fluorescence microscope (Olympus).

The procedure of cell migration assay was followed in a previous study [25 (link)]. First, 1 × 10⁴ per well of MSCs were added into Oris seeding stoppers for 24 h of incubation to reach confluency. Next, the stoppers were removed and used as a pre-migration reference (t = 0 h). The post migration was represented at 24 h. Next, 200 μL of a Calcein AM solution (2 μM, Sigma-Aldrich, Burlington, MA, USA) was added to the culture plates and stained for 30 min at the time points of 0 and 24 h. The cells were observed by a Zeiss Axio Imager A1 fluorescence microscope (White Plains, NY, USA). Cell migration distance was semi-quantified using Image J 5.0 software (Becton Dickinson, Canton, MA, USA). The MSC alone represented as the control.

DLD-1 or HCT-116 cells were serum starved for 16 h in a 1%-FBS-medium. Cells were detached with TrypLE Express Enzyme (Gibco). 5x10⁴ cells were resuspended in a 1%-FBS-medium and they were seeded on Fluoroblok 24-well plate permeable inserts, with 8 μm pores (Corning). Following a 20-22h incubation with chemoattractant (10% FBS in RPMI medium), the inserts were transferred in a 2^nd 24-well plate containing a 4 μg/mL Calcein AM solution (Sigma Aldrich). The fluorescence was read on an inverted fluorescence microscopy and migrated cells were counted with ImageJ software.

BEAS-2B cells were treated with fucoxanthin and incubated with 10 ng/mL TNF-α/IL-4 for 24 h. Human monocytic THP-1 cells were purchased from the Bioresource Collection and Research Center (BCRC, Taiwan) and cultured in RPMI 1640 medium. The THP-1 cells were treated with calcein-AM solution (Sigma) for 30 min as described previously [26 (link)]. We co-cultured the THP-1 and BEAS-2B cells and detected the adherent THP-1 cells using fluorescence microscopy (Olympus, Tokyo, Japan).

DiFi cells were plated in 96-well microplates for fluorescence assays and incubated overnight for their attachment to the plate surface. Calcein AM solution (C1359, Sigma Aldrich, St. Louis, MI, USA) was added to each well following manufacturer’s instructions and fluorescent levels were measured by TECAN Spark 10M instrument. LIP levels are inversely correlated with calcein fluorescence.
Deferoxamine mesylate (DFO, Sigma Aldrich, St. Louis, MI, USA) was supplemented at 200 μM to cells to perform the rescue experiment.