Spark multifunctional microplate reader

To determine the integrity of the bacterial membrane, 8-anilino-1-naphthalenesulfonic acid (ANS, 96%, Aladdin, Shanghai, China) was devoted to evaluating the change in outer membrane permeability towards E. coli, due to the outer membrane unique to the Gram-negative bacteria. In contrast, o-Nitrophenyl β-D-galactopyranoside (ONPG, 98%, Yuanye, Shanghai, China) was employed to measure bacterial inner membrane change in the above two species of bacteria. Briefly, after the antibacterial process, each bacterial solution was disposed of a 500 μL ONPG solution (0.75 mol/L in NaH₂PO₄ buffer, pH 7.0). Finally, the yellow supernatant was removed and detected at the absorption of 420 nm (OD₄₂₀) with a SPARK multifunctional microplate reader (Tecan, Austria GmbH, Kärnten, Austria).

The generation of total ROS levels within the bacteria was determined by a 2′, 7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) probe and a ROS Assay Kit (Cat#BL714A, Biosharp, Beijing, China). After irradiation for 10 min, the bacterial suspension was co-cultured with H₂DCFDA (10 μmol/L in PBS) at 37 °C for 30 min, standing in the dark. After that, 150 μL of bacterial solutions was seeded onto a 96-well plate in order and analyzed by a SPARK multifunctional microplate reader (Tecan, Austria GmbH, Kärnten, Austria) at the excitation/emission wavelength of 488/525 nm.

Mouse fibroblasts (L-929, Beijing, China) were used to study the biocompatibility of different samples [21 (link)]. The L-929 cells were cultured in a 37 °C incubator with 5% CO₂ for 24 h and the cell medium was changed every other day. Subsequently, the cytotoxicity towards L-929 fibroblasts was evaluated by methyl thiazolyl tetrazolium (MTT) assays.
Briefly, 500 μL (3.0 × 10⁴ cells/mL) of the L-929 suspension was added onto the surface of various sterilized samples in a 24-well plate, respectively, and co-incubated for 1, 2, and 3 days, ensuring the sample surface was completely immersed by the cell medium. At each time point, the cell medium was removed before 50 μL of MTT (0.5 mg/mL) was added into the well and returned to the incubator for 4 h in darkness. The supernatant was carefully sucked out twice and then dissolved in 150 μL of DMSO, also being shaken, and vibrated for another 15 min. Finally, the supernatant was evaluated at the absorption of 490 nm (OD₄₉₀) on a SPARK multifunctional microplate reader (Tecan, Austria GmbH, Kärnten, Austria) while the cell medium was applied as the control.

The generation of singlet oxygen (¹O₂) from different sample surfaces was related to 1, 3-Diphenylisobenzofuran (DPBF). Firstly, 500 μL of DPBF (5 mmol/L in DMSO) was co-incubated with different samples in darkness for 10 min. After 808 nm of irradiation, the supernatant was collected and measured at the absorption of 420 nm (OD₄₂₀) with a SPARK multifunctional microplate reader (Tecan, Austria GmbH, Kärnten, Austria).

The enhanced ATP assay kit (Cat#S0026, Beyotime, Shanghai, China) was applied to research the bacterial metabolic activity. After irradiation, the already treated bacteria solution was collected and co-cultured with 100 μL of the lysate before being sonicated for 3 min to fully lyse. Lastly, suck out 20 μL of the supernatant and 100 μL of the working solution, inject them into a 96-well black plate together, and then measure the ATP levels through the program “chemiluminescence” via a SPARK multifunctional microplate reader (Tecan, Austria GmbH, Kärnten, Austria).

Caco-2 and NCM460 cells were plated (4000 cells/well) in 96-well plates, polydextrose, fibersol-2, or fermentation broth of F. nucleatum at the designed concentration was added, respectively, and further cultured for 72 h. MTT (KeyGEN BioTECH, Nanjing, China) was used to assay the cell viability by Spark multifunctional microplate reader (Tecan AG, Männedorf, Switzerland).

Blood of different donors was obtained. Throughout these assays RPMI 1640 without phenol red (Gibco) was used. RBCs were washed twice in RPMI and suspensions at 5% haematocrit were prepared in complete medium (RPMI 0.5% albumax) and 100 µL samples were dispatched in round-bottom 96-well culture plates. Yoda1and Jedi2 were prepared in complete medium at double concentration, and 100 µL added to the wells containing 100 µL RBC suspensions in triplicates (2.5% final haematocrit) and mixed by pipetting. Final concentrations for Yoda1 were 10 µM, 5 µM, 2 µM and 1 µM and for Jedi2 1000 µM, 300 µM and 100 µM. Wells with DMSO were prepared for baseline determination (0.1% for Yoda1, 0.5% for Jedi2). Plates were incubated in standard conditions at 37 °C in 5% O₂, 5% CO₂. After 24 h, 2 µL of saponin (15%) were added to selected wells to obtain total lysis. RBCs were pelleted by centrifugation for 3 min at 1800 g and 150 µL of supernatant transferred to flat bottom 96-well plates. Haemoglobin released into the culture supernatant was quantified using a SPARK multifunctional microplate reader (TECAN) at 540 nm. Lysis was calculated after deduction of DMSO treated samples as percentage of total saponin-induced lysis.