96 well cell culture plate

To determine whether the recombinant T4 Rnl1 had an impact on the growth of S. mutans, overnight cultures of WT, WT_B, and WT_T strains were subcultured in BHI until the OD_{600 nm} reached 0.5 and inoculated at a dilution of 1:100 into fresh BHI broth [18 (link)]. For the optical density method, the growth of these planktonic bacteria was monitored using a 96‐well cell culture plate (Corning, NY, USA) incubated at 37°C for 24 hr. The optical density of the cell culture was measured hourly at 600 nm by a microplate reader with each individual well covered with sterile mineral oil and maintained at 37°C. Wells with medium only (without bacteria) were used as blanks.

Splenocytes (1.5×10⁶) were inoculated in a 96-well cell culture plate (Corning, New York, USA), co-cultured with 40 μg/ml wild-type RBD for 29 h or positive stimulant PMA (0.1 μg/ml) (Sigma-Aldrich, Missouri, USA) + Ion (1 μg/ml) (Sigma-Aldrich, Missouri, USA) for 6 h. After stimulation, cells were stained referring to the instruction of Cytofix/Cytoperm W/Golgi Stop Kit (BD Pharmingen, Chicago, USA). Briefly, the 96-well plate was centrifugated (2,000 rpm, 5 min) and washed twice with PBS. Then, the cells were blocked on ice in 50 μl of CD16/CD32 antibody (1:100 in 2% FASC Buffer) (BD Pharmingen, Chicago, USA) for 20 min, and stained with fluorescent-labeled anti-CD3, anti-CD4, and anti-CD8 monoclonal antibody (final concentration: 1:400 in 2% FASC Buffer) (BD Pharmingen, Chicago, USA) at 4°C for 40 min. After washing, cells were treated with the fixation and permeabilization solution (4°C, darkness, 20 min). Then, cells were washed twice with PBS, and treated with 50 μl/well fluorescent conjugated IL-2/TNF-α/IFN-γ antibody (final concentration 1:200 in 2% FASC Buffer) (BD Pharmingen, Chicago, USA) at 4°C for 40 min. After washing, cells were suspended with 150 μl of 2% FASC Buffer and analyzed by flow cytometry (Beckman Cytoflex LX, Florida, USA).

The viability of CFSC-8B cells was quantified by the ability of living cells to reduce the yellow dye MTT to a blue formazan product. CFSC-8B cells (8 × 10⁴ cells/mL) were seeded in a 96-well cell culture plate (Corning Incorporated, USA) and grew to 80%–90% confluence. Then the cells were incubated with Antrodin C (10–200 μM) for 24 h. The viability (% of the control) of cells treated with Antrodin C was calculated as 100% × (absorbance of treated cells/absorbance of control cells).

Macrophage isolation was based on a previously established and reliable method (23 (link), 24 (link)). Briefly, the head kidney was aseptically removed and placed in a sterile 60 mm cell and tissue culture dish (JET BIOFIL, China) containing RPMI 1640 medium (Gibco, USA) supplemented with 1% penicillin/streptomycin (Gibco, USA). The tissue was gently homogenized with a sterile syringe and filtered through a 70 μm cell strainer (Corning, USA) to obtain a single cell suspension. Cell suspension with a 10 mL volume was layered over a density gradient stratification of 54%/31% Percoll (Sigma-Aldrich, USA) and centrifuged at 400 × g for 40 min at 4°C. Cells were collected from the 54%-31% partition level after centrifugation. Cells were washed with RPMI-1640, counted using 0.1% Trypan blue (Solarbio, China), and adjusted to a concentration of 1 × 10⁶ cells per well in RPMI-1640 complete medium containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) (Gibco, USA) in a 96-well cell culture plate (Corning, USA). Non-adherent cells were removed after 24 h, and MФ was washed and cultured in complete medium for further experiments. All procedures were performed at 25°C in a sterile environment.

Twenty-thousand Vero.E6 cells were seeded per well in a 96-well cell culture plate (Corning; 3340) 1 day prior to performing the assay. Serum samples were heat inactivated at 56°C for 1 hour prior to use. Serum dilutions were prepared in 1× minimal essential medium (MEM; Gibco) supplemented with 1% FBS. Each virus was diluted to 10,000 50% tissue culture infectious doses (TCID₅₀s)/mL, and 80 μL of virus and 80 μL of serum were incubated together for 1 hour at RT. After the incubation, 120 μL of virus–serum mixture was used to infect cells for 1 hour at 37°C. Next, the virus–serum mix was removed and 100 μL of each corresponding dilution was added to each well. A volume of 100 μL of 1X MEM were also added to the plates to get to a total volume of 200 μL in each well. The cells were incubated at 37°C for 3 days and then fixed with 10% paraformaldehyde (Polysciences, Warrington, PA) for 24 hours. The next day, cells were stained using a rabbit anti-nucleoprotein antibody (Invitrogen; PA5-81794) as primary antibody and a goat anti-rabbit secondary antibody conjugated to HRP (Invitrogen; 31460). This protocol was adapted from an earlier established protocol [22 (link),35 (link),43 (link)].

M-CSF- and GM-CSF-differentiated cells were harvested as described above. 1.5 x 10⁵ cells/well were seeded in 100 µl medium in a 96-well cell culture plate (Corning, #3799) and incubated for 18 h. Following pre-incubation for 3 h with 20 ng/ml LPS (Alexis, #581-012-L002), cells were stimulated with crystals/particles for 4 h [final concentration: 6.9 µM nigericin (Invivogen, #Tlrl-nic), 0.8 µg/µl silica, 1 x 10⁶S. cerevisiae particles, and 1 µg/µl of all other crystals [cholesterol, t-CPPD, MSU (lot1), MSU (lot2)]. Supernatants were harvested and IL-1β secretion was determined by ELISA analysis.

MTT assay was used to measure cell viability in cultures. Cells were plated at a density of 4000 cells/mL in a 96-well cell culture plate (Corning, Denmark). After administration, 20 μl of .5 mg/ml of MTT (Solarbio, Beijing, China) was added to each well and cells were incubated at 37°C for 4 h. Following that, 150 μl of dimethyl sulfoxide (DMSO) per well was added to solubilize the formazan crystals in viable cells. Optical density (OD) in each well was quantified at 490 nm wavelength using an ELx808 Ultra Microplate Reader (BioTek, Winooski, VT, United States).