Biotek gen5

Cell viability was assessed using CellTiter-Glo (Promega, Madison, Wisconsin, USA). Briefly, 1000 cells were seeded in white, flat-bottom, 96-well plates (Corning, Corning, NY, USA). After 24 h, drugs were added to the medium, and cells were incubated for 72 h. CellTiter-Glo luminescent reagent was added according to the manufacturers protocol, and the luminescence signal measured on a Synergy LX (Agilent, California, USA) with BioTek Gen5 (v3.08). To evaluate if a combination of drugs is synergistic, cells were simultaneously treated with varying concentrations of drugs, and cell viability was measured with CellTiter-Glo. Synergism scores were obtained using the R package SynergyFinder (v2.2.4)^{75 (link)}.

H460 parental cells and two CD109 deficient cell clones were grown to confluence in five different experiments. Cells were harvested and total RNA was isolated from the cells using the NucleoSpin RNA Kit (#740955.50, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) following the manufacturer’s specifications. Quality control on concentration and integrity of the isolated RNA was performed with the BioTek Epoch, and its Take3 Microvolume Plates, and the software BioTek Gen5 (Version 3.14, Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instructions. RNA libraries were prepared for sequencing using the TruSeq Stranded mRNA protocol (Illumina, Inc., San Diego, CA, USA). Post-library quality control included evaluation of library concentrations and fragment sizes (100–400 bp) on a LabChip. All libraries were index barcoded, enabling multiplexed sequencing. All samples were sequenced on the same lane of a flow cell on a NovaSeq 6000 sequencing system (Illumina, Inc., San Diego, CA, USA) with 2×50 bp.

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)).