Biotek gen5

To investigate the effects of AlNPs on the tightness of BBB, we assessed the permeability rate of 40 kDa FITC–dextran (Sigma-Aldrich) with or without treatment of AlNPs. Upon the completion of AlNP treatment, we added 10 μg/mL of 40 kDa FITC-dextran to L.C. of hNVUI and collected the conditioned medium from R.C. side every 6 h up to 24 h. We measured the concentration of BBB-penetrating FITC-dextran by Bio Tek Gen5 (Agilent Technologies, Santa Clara, CA, USA). The apparent permeability of each sample across the BBB (P_BBB) was calculated by Eq. 1 as described previously^{15 (link),46 (link)}: $${\text{P}}_{\text{BBB}}=\frac{C_L}{C_0}\times \frac{V}{S·t}$$
In this equation, C_L (μg/mL), C₀ (μg/mL), V (cm³), S (cm²), and t (s) represent the concentration of FITC-dextran in R.C., the initial concentration of FITC-dextran in L.C., the volume of R.C., the surface area of microchannels connecting L.C. and R.C., and the time for BBB penetration, respectively.

Bacopa extracts’ toxicity was determined by assessing cell viability in the RAW 264.7 cells through the MTT assay described by Barbosa et al. [24 (link)]. After the incubation period (35,000 cells/well, 24 h at 37 °C), the RAW 264.7 cells were incubated for 45 min with MTT (0.5 mg/mL prepared in DMEM), which was converted by dehydrogenases of metabolically active cells from a yellow salt into an insoluble purple formazan product. The pellet was further solubilized with 100 µL DMSO and the absorbance measured at 510 nm in a microplate reader (Biotek, Bad Friedrichshall, Germany) operated using Biotek Gen 5™ software.

Uteri were harvested from CBAF1, Tlr4^+/+, and Tlr4^−/− female mice identified as estrous by vaginal lavage cytology, and epithelial cell monolayers (> 90% epithelial cells) were prepared as previously described^{13 (link),79 (link)}. Enriched epithelial cells were resuspended at 7.5 × 10⁵ cells/ml in DMEM + FCS, and 150 μl aliquots were plated in 48-well multidishes (Nunc, Roskilde, Denmark). Cells were incubated for 4 h at 37 °C in 5% CO₂ to permit adherence, then sperm (3.8 × 10⁵, 7.5 × 10⁵, or 1.5 × 10⁶ /well to give 1:2, 1:1, and 2:1 sperm to uterine epithelial cell ratio), seminal vesicle fluid (1:2 dilution of 10% seminal vesicle fluid in DMEM + FCS), or a combination of both sperm and seminal vesicle fluid (5% seminal vesicle fluid and 1.5 × 10⁶ sperm/well) were added to a final volume of 200 μl. Supernatants were collected after 16 h incubation, and following replacement of culture medium and a further 24 h incubation, then centrifuged at 400 × g to remove cellular debris, and stored at −80 °C until cytokine assay. Adherent cells were quantified by Rose Bengal dye uptake (0.25% in PBS, 10 min at room temperature) (Sigma-Aldrich) and cell lysis in 0.1% SDS by measuring absorbance at 570 nm using a BioTek Synergy H1 Hybrid Multi-Mode Reader and BioTek Gen5 software (BioTek Instruments, Inc., Winooski, VT, USA) as described previously^{13 (link),79 (link)}.

The levels of LDH in supernatant medium were measured using CyQUANT™ LDH Cytotoxicity Assay (Invitrogen; catalog no. C20301) according to the manufacturer's instructions; the LDH value measured in nontreatment using the lysis buffer was set to 100% of the cytotoxicity. The level of LDH was measured with a BioTek Cytation™ 5 Cell Imaging Multi‐Mode Reader and analyzed using BioTek Gen5 software (BioTek Instruments Inc.).

Knee joint tissue sections were then embedded in paraffin and stained with hematoxylin and eosin (H&E) and safranin O (SAF O) according to the manufacturer's instructions. Images of the stained knee joint tissues were captured with a BioTek Cytation 5 Cell Imaging Multi‐Mode Reader and analyzed by BioTek Gen5 software (Bio‐Tek Instruments Inc.). H&E‐stained images were used for inflammation scoring and to characterize synovial hyperplasia and synovial cellularity according to previous guidelines (Kwon et al, 2021 (link)). SAF O‐stained images were used to generate OARSI scores and to characterize osteophyte formation, proteoglycan depletion, bone erosion, and bone formation according to previous guidelines (Kwon et al, 2021 (link)).

Cell viability was measured using RealTime‐Glu™ MT Cell Viability Assay (Promega Corporation, WI, USA; catalog no. G9711) according to the manufacturer's instructions. The luminescence signal was measured using a BioTek Cytation™ 5 Cell Imaging Multi‐Mode Reader and analyzed using BioTek Gen5 software (BioTek Instruments Inc.) at intervals of 30 min for 12 h total under a humidified atmosphere containing 5% of CO₂ at 37°C.

Different concentrations of DMF (140, 420, and 700 μg/ml) and vancomycin (2.5 mg/ml) were added to LB (200 μl; Invitrogen) medium containing oxacillin (6 μg/ml; Sigma‐Aldrich Co.) in 96‐well plates (BD Biosciences). MRSA (4 × 10⁶ CFU) was seeded into the medium and incubated for 24 h; DMSO was used as the negative control. MRSA (4 × 10⁶ CFU) seeded into LB (200 μl; Invitrogen) medium containing oxacillin (6 μg/ml; Sigma‐Aldrich Co.) in 96‐well plates (BD Biosciences) and incubated at 35°C for 4 h. Different concentrations of DMF (140, 420, and 700 μg/ml) were applied to medium for 20 h; DMSO was used as the negative control.
The media was removed and slides were fixed with methanol (J.T. Baker, Center Valley, PA, USA; catalog no. 909302) for 10 min and then washed with DPBS (Thermo Fisher Scientific, Inc.) thrice. 200 μl of crystal violet solution (0.1%; Ward's Natural Science, NY, USA; catalog no. 548‐62‐9) was then applied and allowed to incubate for 15 min, after which the slides were washed with DPBS (Thermo Fisher Scientific, Inc.) thrice. 200 μl of acetic acid (33%; Sigma‐Aldrich Co.; catalog no. 695092) was added, and absorbance (570 nm) was measured using a BioTek Cytation™ 5 Cell Imaging Multi‐Mode Reader and analyzed using BioTek Gen5 software (BioTek Instruments Inc.).