96 well u bottom plate

Compounds were
serially diluted 2-fold from stock solutions (1.0 mM) to yield 12
100-μL test concentrations, wherein the starting concentration
of dimethyl sulfoxide (DMSO) was 2.5%. Overnight S. aureus, E. faecalis, E. coli, A. baumannii, USA300-0114 (CA-MRSA), and ATCC 33591 (HA-MRSA)
cultures were diluted to ca. 10⁶ CFU/mL in MHB or TSB and
regrown to midexponential phase, as determined by optical density
recorded at 600 nm (OD₆₀₀). All cultures were then diluted
again to ca. 10⁶ CFU/mL, and 100 μL was inoculated
into each well of a U-bottom 96-well plate (Corning, 351177) containing
100 μL of compound solution. Plates were incubated statically
at 37 °C for 72 h, upon which wells were evaluated visually for
bacterial growth. The MIC was determined as the lowest concentration
of compound resulting in no bacterial growth visible to the naked
eye, based on the highest value in three independent experiments.
Aqueous DMSO controls were conducted as appropriate for each compound.

Influenza virus titers in mouse lung tissues were determined using the tissue culture infective dose 50% (TCID50) combined with hemagglutination (HA) assay. Mouse lungs were placed in 2 ml of ice-cold S and homogenized using gentleMACS Dissociator (Miltenyi Biotec GmbH) and centrifuged at 600 g for 10 minutes. Supernatant was collected and used for the TCID/HA assay. Ten-fold serial dilutions of the supernatants were prepared using Minimal Essential Medium (MEM, ATCC) containing 2 μg/ml TPCK. MDCK (1.0×10⁵ cells/mL) cells were plated in a U-bottom 96-well plate (Corning). The diluted supernatants were added to the 96 well plate and then incubated overnight at 37°C/ 5% CO₂ incubator. The next day, the medium was replaced with fresh MEM. After 72 hours of incubation, 50 μl of 0.5% turkey RBCs in S was added to each well. The plates were then incubated for 1 h at 4°C and the HA patterns were read to determine the TCID50. A positive (PR8) and corresponding negative controls were added in each plate. Hazy wells show turkey erythrocytes agglutination while non-agglutinated wells show a distinct RBC “button” at the bottom of the well. TCID50 was then calculated by the Reed-Muench formula [33 , 34 ].

The protocol for cell labelling and antibodies for targeting bovine immune cells were previously described (42 (link)). Briefly, cryopreserved PBMCs and PP cells were re-suspended to a concentration of 2 x 10⁷ cells/ml in cell-labeling buffer (PBS containing 0.2% w/v gelatin and 0.03% v/v sodium azide), and a 50-µl aliquot added to each well of a U-bottom 96-well plate (Corning, Inc.). An equal volume of primary monoclonal antibody (Supplementary Table 2) diluted in cell-labeling buffer was added to each well, incubated on ice for 20 min, and washed three times with cell-labeling buffer. Fluorochrome-conjugated anti-Ig isotype-specific antibodies (Supplementary Table 2) were added to each well, incubated on ice in the dark for 20 min, and washed three times in cell-labeling buffer. Cells were fixed using 2% formaldehyde in PBS, and stored at 4°C in the dark. Samples were analyzed with a FacsCalibur (Becton-Dickinson; NJ) using CellQuestPro acquisition and analysis software. A minimum of 10,000 events were captured for each sample and data was collected in list-mode. Isotype controls (Supplementary Table 2) and unstained cells were used to monitor the specificity of the cell staining protocol and reagents, and used to set gates for quantifying specific cell populations.

Culture samples frozen at −20°C in 96‐well plates were thawed in a 42°C water bath for 30 min. Sample plates were then centrifuged at 4,255 g for 10 min three times, each time pipetting half the supernatant into a clean plate. After the third centrifugation, 100 μl of supernatant was pipetted into a U‐bottom 96‐well plate (Corning #3797). This plate was covered with an AluminaSeal (Diversified Biotech #ALUM‐1000) and kept at 4°C until analysis. Supernatant analysis was performed using an Agilent 1260 Infinity Liquid Chromatography system with an inline Aminex HPX‐87H column (#125‐0140) and Micro‐Guard Cation column (Bio‐Rad #125‐0129) running a 5 mM sulfuric acid mobile phase at 0.6 ml/min. Purified supernatant samples in 96‐well plates were placed in an autosampler cooled to 4°C. Following sampling of 10 μl, the autosampler needle was cleaned with a 3‐s rinse of 10% isopropanol. The peaks for glucose (9.2 min) and acetate (15.5 min) were detected with a Refractive Index detector (RID; Agilent #G1362A). Both the columns and the RID were heated to 35°C. Standard curves of glucose (Sigma #049K6201) and acetate (Fluka #57191) were run to enable quantification. Integration of the RID peaks for glucose and acetate was done automatically in Chemstation software (Agilent).

F49B7 retinal organoids were infected at week 35 with AAV (serotype, PhP.eB; Addgene, #67526) inducing the expression of the calcium sensor GCaMP6s under the control of the generic promoter EF1α.
Individual organoids were placed in a single well of an ultra-low attachment U-bottom 96-well plate (Corning, #7007) and maintained at 37°C in 5% CO₂ in 27 μl culturing media and 3 μl AAV. After 5 hours, 70 μl of fresh media was added to each well. One day later, 100 μl of fresh media was added to each well. After 24 hours, and every 48 hours thereafter the solution was completely exchanged with fresh media.

The TCR-transduced T cells were incubated in a U-bottom 96-well plate (Corning) with 1 × 10⁵ cells/well in a ratio of 1:1 with T2 cells loaded with different concentrations of NS3 and NS5 peptides (1,000, 100, 10, 0.5, 0.1, and 0 ng/ml). T2 cells loaded with HIV peptide (1,000 ng/ml) were included as a negative control. After 6 h of coculture, the intracellular content of human IL-2, TNFα, IFNγ, and CD107a in the TCR-transduced T cells was evaluated by intracellular multicolor flow cytometry staining.