br For cell culture in the CDEAC
For cell culture in the CDEAC hydrogel, 10 wt% Eu(III) complex-CMC and K-DPY-CMC suspension in water were sterilized by UV light for 20 min. The Eu(III) complex-CMC was mixed with sterilized deio-nized water and concentrated PBS (10 × ). Subsequently, the cell sus-pension was added. The final CMCs concentration in the mixed sus-pension was 1.0, 2.0, 3.0, or 4.0 wt%. The resulting mixed suspension at a cell density of 1 × 105 cells/mL was transferred into a 12-well plate (300 μL/well) and incubated at 37 °C for 30 min. Then, 100 μL of DMEM medium and K-DPY-CMC with the same concentration as Eu(III) complex-CMC in the mixed suspension were added to the wells. The plates were incubated with 5% CO2 at 37 °C. The cell culture medium was changed every 3 days.
A methyl thiazolyl tetrazolium (MTT) assay was performed for in
Fig. 2. (A) Emission spectra of Eu(III) complex-CMC (black line), K-DPY-CMC (Blue line) and CDEAC (red line) in water solution (ex = 274 nm). (B) Emission spectra of CDEAC (2 wt%, ex = 274 nm) with incremental addition of 0–2 μM ClO− in water. (C) Emission spectra of Eu(III complex-CMC-ClO- (2 wt% CDEAC and 2 μM ClO−, ex = 274 nm) upon addition of 0–10 nM SCN− in water. (D) Fluorescence intensity of CDEAC during 5 cycles of adding ClO− and SCN−. (E) Schematic illustration of the responsive mechanism of CDEAC after adding ClO− and SCN−. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
vitro cytotoxicity of CMC, The CMC suspension was mixed with ster-ilized deionized water and concentrated HBSS (10 × ). Subsequently, the MCF-7 cell suspension was added. The final CMC concentration in the mixed suspension was 1.0, 2.0, 3.0 or 4.0 wt%. The mixed sus-pension was seeded into a 96-well plastic flask with a density of 5 × 103 3X FLAG Peptide per well in DMEM with 10% FBS at 37 °C under 5% CO2 and incubated for 48 h. Thereafter, MTT (10 μL, 5 mg/mL) was added to each well and the plate was incubated for 4 h at 37 °C under 5% CO2. After the addition of dimethyl sulfoxide (DMSO, 100 μL per well), the plate was allowed to stand at 37 °C for 15 min. The optical density was measured at 490 nm using a microplate reader (Wallac 1420 Victor).
2.8. Spheroid release from CMC hydrogels
To release CSs, the CMC hydrogel was maintained at 10 nM ClO− for 15 min to transform it into a suspension. 200 μL of HBSS was then added to each well containing a mixed suspension of CSs and CMC. The suspension of CSs and CMC was gently aspirated and collected into a
centrifuge tube. Subsequently, 2 mL of HBSS was added into the cen-trifuge tube and the suspension was centrifuged at 800 rpm for 1 min, after which the supernatant was removed.
After 15-day culture of cells in the CDEAC hydrogels, a solution of Calcein AM (Invitrogen) and Ethidium Homodimer I (Invitrogen) in HBSS was added to the wells containing CS-laden CDEAC hydrogels. The CSs were stained for 20 min at room temperature and imaged by Leica DR confocal microscope equipped with a digital camera (ORCA-ER, Hamamatsu) at room temperature. For the live/dead assay of CSs released CSs, the procedure was identical.
2.10. Confocal microscopy of CSs
After cultured and/or released from the CDEAC hydrogels as de-scribed above, CSs were fixed with 400 μL 4% paraformaldehyde
Fig. 3. (A) Photograph of CDEAC hydrogel formation. (B) Bright field and fluorescence images of CDEAC hydrogel in response to ClO− and SCN−.
diluted in PBS in a 12 well-plate. The CSs were allowed to submerge in this solution for 20 min at room temperature. Subsequently, they were washed three times with 1 mL PBS for 5 min 1 mL of methanol was then added to permeabilize the cells for 10 min and then washed three times with PBS. 500 μL of antibody (Alexa Fluor® 488 E-Cadherin Rabbit monoclonal antibody or Alexa Fluor 488-labeled Goat Anti-Rabbit IgG and Alexa Fluor-568 Phalloidin, Cell Signaling Technology) or cell membrane probe Dio was added into the 12 well-plate, which was al-lowed to incubate for 20 min. To remove excess antibody and probe, the CSs were rinsed three times with 1 mL of PBS at room temperature. To stain cell nuclei, 0.5 μg/mL Hoechst (Invitrogen) in PBS was added into the 12-well plate containing CSs for 20 min at room temperature. The structure of CSs was visualized and imaged using a Leica DR confocal microscope. Pictures were processed using Photoshop software (Adobe Systems, CA).