Flat bottom 96 well microtiter plates

Biofilm assays were performed based on previously published protocols 14 (link)52 (link), with minor modifications. Briefly, overnight fungal cultures were washed three times in PBS and resuspended in Dulbecco's modified eagle medium (DMEM, Gibco) at a final concentration of 10⁶ cells ml^-1. For some experiments, DMEM was supplemented with 1 mM, 50 mM, or 100 mM LiCl, 100 mM NaCl, 20 µg ml^-1 EBS, or a combination of these chemicals. Next, 200 µl of fungal cells were transferred into individual wells of sterile, polystyrene, flat-bottom, 96-well microtiter plates (Corning). Wells with medium only were included as controls. To minimize evaporation, 200 µl sterile water was added to the wells surrounding the test wells. Plates were incubated at 37°C and 5% CO₂ for 48 h.

Spleen cells, prepared as described previously, were suspended in RPMI medium (Sigma Chemicals) supplemented with 10% fetal calf serum, 10 mM HEPES, 2 mM l-glutamine, 1 mM sodium pyruvate, and 50μM β-mercaptoethanol. They were distributed in flat-bottom 96-well microtiter plates (Corning) at a density of 5 × 10⁵ cells per well, in 200 μL of culture medium alone or medium with STAg (10 μg/mL) in triplicate. Cells were stimulated with concanavalin A (2 μg/ml) as positive control. The plates were incubated for 3 days in atmosphere containing 5% CO₂ at 37°C and pulsed with 1 μCi [³H]thymidine per well for an additional 18-h period. The cells were next harvested onto glass fiber filters and incorporated radioactivity (counts per minute [cpm]) was measured in a liquid scintillator. The results are expressed as mean counts per minute (count) for three replicates.

Flat bottom 96-well microtiter plates (Corning) were coated with 300 ng of SARS-CoV-2 spike protein per well diluted in 100 μl of ELISA coating buffer (1XPBS, 5.3g Na₂CO₃, 4.2g NaHCO₃, pH 9.6) and incubated overnight at 4 °C. The next day, spike protein was removed and 125 μl of 5% BSA blocking buffer were added to each well and incubated for 1 hour at 37 °C. SARS-CoV-2 spike protein coated and blocked plates were then wasted plate three times with 200 μl PBST. Serum samples were subjected to thermal inactivation after being initially diluted in a BSC. Inactivated serum samples were then serially diluted 2-fold in PBS. One hundred microliters of each dilution was added in duplicate to the antigen coated plate, sealed, and incubated at 37°C for 1 hour. Plates were then washed three times with 200 μl PBST. One hundred microliters of secondary antibody, anti-human (H&L) HRP conjugated, diluted 1:5000 in PBS to each well was added to each well and plates were incubated at 37°C for 1 hour. Plates were then washed three times with 200 μl PBST. Eighty-five microliters ABST Substrate Solution (Thermo Fisher Scientific) was added to each well and plates were agitated (900 rpm) at room temperature for 30 minutes, then analyzed at 650 nm absorbance on a plate reader (Molecular Devices, San Jose, CA).

Biofilms on microscope slide were grown as described previously [19 (link)]. For biofilm formation on a polystyrene surface, flat-bottom 96-well microtiter plates (Corning Inc.) were used [18 ]. E. coli overnight cultures were diluted 1:40 in fresh medium, and 150-μL aliquots were dispensed into wells. After 24 h of incubation (37 °C), cell density was measured (OD₆₀₀) using a plate reader, and 30 μL of Gram Crystal Violet (Remel) was applied for staining for 1 h. Plates were washed with water and air dried, and crystal violet was solubilized with an ethanol-acetone (4:1) solution. The OD₅₇₀ was determined from this solution, and the biofilm amount was calculated as the ratio of OD₅₇₀ to OD₆₀₀ [19 (link)].

Flat bottom 96-well microtiter plates (Corning) were coated with 300 ng of SARS-CoV-2 spike protein per well diluted in 100 μl of ELISA coating buffer (1XPBS, 5.3g Na₂CO₃, 4.2g NaHCO₃, pH 9.6) and incubated overnight at 4 °C. The next day, spike protein was removed and 125 μl of 5% BSA blocking buffer were added to each well and incubated for 1 hour at 37 °C. SARS-CoV-2 spike protein coated and blocked plates were then washed three times with 200 μl PBST. Serum samples were subjected to thermal inactivation after being initially diluted in a BSC. Inactivated serum samples were then serially diluted 2-fold in PBS. One hundred microliters of each dilution was added in duplicate to the antigen coated plate, sealed, and incubated at 37 °C for 1 hour. Plates were then washed three times with 200 μl PBST. One hundred microliters of secondary antibody, anti-human (H&L) HRP conjugated, diluted 1:5000 in PBS to each well was added to each well and plates were incubated at 37 °C for 1 hour. Plates were then washed three times with 200 μl PBST. Eighty-five microliters ABST Substrate Solution (Thermo Fisher Scientific) was added to each well and plates were agitated (900 rpm) at room temperature for 30 minutes, then analyzed at 650 nm absorbance on a plate reader (Molecular Devices, San Jose, CA).

Overnight cultures were diluted to OD₆₀₀ ~0.05 and grown in TSB at 37 °C with rotary shaking at 180 rpm. Aliquots (100 μL) were inoculated into flat bottom 96-well microtiter plates (Corning, Corning, NY), and bioluminescence was detected using a BioTek Synergy Neo2 plate reader (Agilent, Santa Clara, CA).