Tcm 400

To evaluate the calcium phosphate deposits of hMSCs, alizarin red was monitored. hMSCs were seeded in 24-well plates with a cell concentration of 5 × 10³ cells/mL. The basal α-MEM was used as the negative control. For positive control, an osteogenic medium was prepared by supplementing the basal medium with 10 nM of dexamethasone (DEX), 10 mM of β-glycerophosphate, and 100 µg/mL of L-ascorbic acid. The monolayer of hMSCs was exposed to media containing NPs at a concentration of 200 µg/mL (cut-off-level particle concentration with no cytotoxic). The cell culture media containing NPs were regularly replaced, twice a week, over a period of 3 weeks. During the 1st, 2nd, and 3rd weeks in culture, the cells were immobilized with 4% paraformaldehyde. The calcification of hMSCs was identified by employing 2% Alizarin Red S solution in deionized water at pH 4.2. Images were captured utilizing an inverted optical microscope (LABOMED TCM400, Los Angeles, CA, USA) with the ToupView program. To conduct the quantification assay, the stained cells were rinsed with deionized water to eliminate nonspecific staining. The stain was extracted using 10% (w/v) cetylpyridinium chloride in 10 mM of sodium phosphate buffer at pH 7.0, and absorbance was subsequently measured at 562 nm using a microplate reader (Infinite^® 200 Tecan, Grödig, Austria).

To evaluate calcification leftovers in the microcysts, the slides were deparaffined and the watering steps were performed. The slides were stained with Alizarin solution (Merck −1.06278) for 1–5 min and observed under light microscope. Calcium red-orange color usually appears in 2 min. To remove excess paint, the slides were first placed in 100% acetone and then in acetone-xylene solution in a one-to-one ratio. After clarification steps, photography was performed with a light microscope (TCM 400, Labomed Inc., CA) (28 (link)).

An inverted fluorescence microscope (TCM400, Labomed Co., USA) equipped with a digital camera and a 4× objective lens was used to record the droplet generation process (see Movie S1). After the thermal cycling process, microdroplets were transferred into an observable chamber via PTFE tubing. A 10× objective lens was used to capture the fluorescence images with a higher resolution. Additionally, the excitation light and the EvaGreen emission were filtered by a B-excitation filter. By utilizing the EvaGreen dye-based Master Mix, the PCR amplification was analyzed by fluorescence intensity of droplets instead of gel electrophoresis. An image analysis software (ImageJ, USA) was used to detect the fluorescence intensity of the droplets in the positive and negative controls, and the results were analyzed using the equation of Poisson distribution: $$P_k=\frac{{\lambda }^k{e}^{-k}}{k!}$$
where $P_k$ is the probability of encapsulating k DNA molecules in one droplet, and $\lambda$ refers to the average number of DNA molecules per droplet which could be calculated using the concentration of stock DNA template ( $C_2$ ) obtained from Eq. (1), mean volume of droplets ( $V_d$ ), and a dimensionless number representing the dilution factor ( $D$ ): $$\lambda ={\mathit{DC}}_2{V}_d$$

The spheres were collected by centrifugation and digested to single-cell suspensions using Dispase II (20U/mL of DMEM). After passing through a 40 μm filter, 250 cells/cm² cells in 10 mL of medium was added to non-adherent (0.1% agarose coated) or adherent culture flasks for daughter sphere formation and colony formation, respectively. The floating spheres and colonies were observed using phase contrast microscope (Labomed TCM 400). The viability of the cells was also confirmed by Trypan blue exclusion method.

We used a HeLa cell line that was obtained several years ago for human cervical adenocarcinoma (CCL-2™; ATCC). HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM D6429; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (12103C; Merck-Millipore), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (P4083; Sigma-Aldrich) at 37°C, 5% CO₂ in a humidified atmosphere. Cells (5 × 10⁴ cells/well) were cultivated in 96-well plates [19 (link)]. The cells were observed, and images were acquired (magnification, 20×) using an inverted microscope (Labomed TCM 400) and then separated by centrifugation at 600 × g for 6 min. The supernatant was gently removed, and the cell pellet was counted. Three replicates were used for each treatment.

To evaluate the mineralization nodules in vitro of MC3T3-E1 cells, alizarin red S staining was used. MC3T3-E1 cells were seeded and cultured in the scaffolds with a cell concentration of 1 × 10⁴ cells/mL. Cell culture media were changed twice a week for 14 days. After 7 and 14 days in culture, the cells were fixed with 4% paraformaldehyde for 30 min. Then, the fixed cells were stained with 2% alizarin red S in PBS at pH 4.2 to detect calcified tissue formation. Images were obtained with an inverted optical microscope (LABOMED TCM400) using ToupView program.