Ts100 microscope

Two adherent cell lines—murine L929 fibroblast (ATCC^® CCL-1™, American Type Culture Collection, Manassas, VA, USA) and MCF-7 human breast adenocarcinoma (ATCC^® HTB-22™, American Type Culture Collection, Manassas, VA, USA)—were chosen for cytocompatibility studies of the h-BN_AuNP nanocomposite.
For morphology analyses (phase contrast and holographic microscopy), cells of each line were seeded into T25 flasks (Sarstedt, Nümbrecht, Germany) and maintained in standard cell culture conditions at 37 °C, 5% CO₂, 95% humidity. Complete Dulbecco’s Modified Eagle Medium (DMEM) culture medium supplemented with 10% heat inactivated fetal bovine serum (FBS) (PAA Laboratories GmbH, Pasching, Austria), 2 mM l-glutamine, 50 IU mL⁻¹ penicillin and 50 µg mL⁻¹ streptomycin (Sigma-Aldrich, St. Louis, MO, USA) was used in the study.
For cytocompatibility analysis, the cells were seeded into 96-well plates (Corning Inc., New York, NY, USA) and cultured for 24, 48 and 72 h in standard conditions mentioned above. All cell cultures were monitored with a Nikon TS-100 microscope (Nikon, Melville, NY, USA).

The experimental groups were added with pyrotinib (20% IC50, 50% IC50), SHR6390 (20% IC50, 50% IC50), and the combination of the two drugs (50% IC50 pyrotinib and 50% IC50 SHR6390), and the no-drug group was the blank control group. The six groups of breast cancer cell lines were cultured in six-well plates. When the cell fusion degree was 80%, the cells were scratched with a 200-μl sterile pipette tip, washed twice with PBS, and added with serum-free medium for culture. Then, we used a Nikon TS100 microscope (Nikon, Tokyo, Japan) and randomly selected in each well three fields of view (×100) in the scratched area to take pictures at 0 h. The medium containing the drug and 10% fetal bovine serum was added, and after 24 h, each well was photographed in the abovementioned field of view. Measure the width of the scratch at different times, compare the difference in scratch healing between the groups, perform the test at least three times, and take the average value (Justus et al., 2014 (link)).

DNA damage was assessed using the OxiSelect DNA Double Strand Break (DSB) staining kit (Cell BioLabs, San Diego, CA). N9 microglia were plated in black-walled 96-well plates at a density of 10,000 cells/well. The following day the cells were exposed to 0, 50, or 100 mJ/cm²∙min for 6 hours using the 96-LED device with half of the wells for each light dosage receiving etoposide (100 uM; Cell BioLabs) during the last 1 hour to serve as positive controls. Cells were then immediately fixed and stained as per kit instructions. Fluorescent staining was visualized on a Nikon TS-100 microscope equipped with a B-2E/C filter cube (Nikon, Melville, NY) with illumination provided by a 120 W light source (XCite 120 lamp; Lumen Dynamics, Mississauga, ON, Canada). Images were captured using a Nikon DS-L1 camera system; illumination intensity and exposure time were kept constant across all images.

The bacterial agglutination experiment was carried out according to previous study with some modifications [47 (link)]. V. parahaemolyticus were cultured to the logarithmic phase and harvested by centrifugation at 2000× g for 10 min. The pellets were re-suspended with sterilized PBS at a density of 1 × 10⁸ cfu/mL after washing with PBS three times. The bacteria were labeled with 0.1 mg/mL fluorescein isothiocyanate (FITC) and slowly shaken overnight in the dark. Then, the FITC-labeled bacteria were rinsed with PBS and re-suspended in PBS at a density of 1 × 10⁷ cfu/mL and mixed with an equal volume of 1 mg/mL rLvCD14L. The PBS and rTrx were used as negative controls. Each treatment was performed in triplicate. After incubation at room temperature for 1 h, the treated cells were observed under an optical Nikon TS100 microscope (Nikon, Tokyo, Japan).

Fibronectin and octadecyl rhodamine B chloride (R18) were obtained from Sigma-Aldrich (St. Louis, MO, United States) and Invitrogen (Carlsbad, CA, United States), respectively. IL-1β, IL-6, and TNF-α were obtained from Sino Biological (Beijing, China). Adhesive substrates were prepared from perfusion of microfluidic channels in the BioFlux plate with different concentration of FN at 5 dyn/cm² for 15 min, followed by incubation at RT for 1 h, and blocked with 0.5% BSA at 5 dyn/cm² for 15 min. All cytokine-containing adhesive substrates were prepared from perfusion of cytokines together with FN. MSCs were trypsinized, stained with 300 nM R18, resuspended at 1 × 10⁶ cells/ml in PBS, and perfused from the inlet wells for 10 min at 10 dyn/cm². In experiments that assess the effect of soluble cytokines, cells were incubated with different cytokines for 30 min at RT before perfusion. Images of attached cells after the perfusion were captured at a magnification of 100× using a Nikon TS100 microscope (Nikon Instruments, Inc., Melville, NY, United States) equipped with a CCD camera (QICAM, QImaging, Surrey, British Columbia) and the BioFlux 200 software. Cell number from each image was counted and averaged for each group.

The Gram-negative bacteria (E. coli and V. harveyi), and Gram-positive bacteria (S. epidermidis) were cultured overnight. Then, bacteria cells were diluted to 10⁵ cfu/mL. After that, 64 μM LBDv peptide solution was incubated with the bacteria at room temperature within 30 min. The PBS and 64 μM pGFP peptide solution were used as negative control. The cells were then added to the glass slide and observed under optical Nikon TS100 microscope (Nikon Corporation, Tokyo, Japan).