Black 96 well plate

Primary macrophages were harvested from four mice per strain, by intraperitoneal lavage four days following intraperitoneal injection of 1.5ml 4%Thioglycollate (BD, Sparks, MD). All mice were injected with the same batch of thioglycollate. Cells from each strain were pooled, and plated in replicate wells (n≥ 4) of 96-well black plates (Fisher, Pittsburgh, PA) at a cell density of 3×10⁵ cells per well in DMEM with 20% FBS at 37°C and 5% CO₂. After overnight culture, cell media was replaced with 1% FBS DMEM media for controls or with media plus 10μg/mL DiI-acetylated LDL (Biomedical Technologies, Ward Hill, MA), or media plus 10μg/mL DiI-acetylated LDL and 200μg/mL unlabeled acetylated LDL (Biomedical Technologies). Four hours later, these media were removed and wells were washed 3 times with PBS and measured for DiI fluorescence (Excitation at 530 nm; Emission at 590 nm).

The AAMCA (7-amino-4-methyl Coumarin-Arachidonamide) hydrolysis assay was performed in 96-well black plates (Nunc) in a total volume of 100 μl. To begin, 0.5 μg microsomal protein (in 50 μl), prepared as described above, was incubated with or without the designated inhibitor in assay buffer (50 mM HEPES, 1 mM EDTA (pH7.4) and 1.4 mg/ml bovine serum albumin (BSA, 0.1% final concentration)) for 30 min at room temperature. Immediately following the incubation, 1 μM AAMCA (Cayman Chemical)substrate prepared in assay buffer was added to the microsomal protein, the components were shaken for 2 minutes and measured kinetically for 60 min at 37°C on a Perkin Elmer VICTOR Nivo (PerkinElmer, Waltham, MA) microplate reader. Pure AMC (7-amino-4-mthylcoumarin, Cayman Chemical) was used to generate 0, 20, 40, 60, 80, and 100 pmol/well standard curve of AMC standard in assay buffer. Values were corrected for background fluorescence observed from well containing buffer alone.

catalase activity in plasma samples was measured with the catalase fluorometric detection kit (ENZO Life Sciences, Lörrach, Germany), and the procedure was optimized according to the manufacturer’s recommendations. Plasma samples (diluted 50-fold) or standard curve samples were aliquoted into 96-well black plates (NUNC, Roskilde, Denmark). A standard curve was derived by diluting an appropriate amount of catalase (Sigma Aldrich, Saint Louis, MO, USA) in 1X reaction buffer which was applied to the plate. Then, 50 μL of 40 μM H₂O₂ solution (ENZO Life Sciences, Lörrach, Germany) was added to each well, and the plate was incubated for 45 min at room temperature. A further 100 µL of the Reaction Cocktail (ENZO Life Sciences, Lörrach, Germany) was added to each well, and the plate was incubated in the dark for additional 10 min. After incubation, all samples were analyzed in the Tecan Infinite 200 plate reader (Tecan, Grödig, Austria) at an excitation wavelength of 570 nm. Fluorescence was measured at 600 nm. A standard curve was produced by plotting relative fluorescence units against catalase activity (U/mL) in each standard sample used for the measurement. The concentration of unknown samples was determined via interpolation to the standard curve. The standard curve ranged from 0 up to 4 U/mL.

Macrophages at a concentration of 1 × 10⁶ cells per well were placed in tetraplicates on 96-well black plates (Nunc, Roskilde, Denmark) at a volume of 200 μL of RPMI1640 with 10% fetal calf serum (FCS, Gibco Life Technologies, Grand Island, NY, USA), and incubated with either luminol or lucigenin (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37 °C. Afterwards, macrophage oxidative burst was stimulated in half of the wells with mouse serum-opsonized zymosan (Sigma-Aldrich, St. Louis, MO, USA) added at a 10:1 ratio (particles per cell) just before the measurement of luminol- or lucigenin-emitted chemiluminescence with a Lucy 1 luminometer (Anthos, Salzburg, Austria), lasting for 75 min. The averaged results of ROI generation were expressed in relative units of luminescence emission (RULE) per second.

To measure monobody stability in serum, 150 μL of Rluc8-fused monobody in PBS (60 μg) was mixed with an equal volume of mouse blood serum and incubated at 37 °C for various incubation times on 96-well black plates (Thermo Fisher Scientific). The bioluminescence activity was measured after adding 10 µL of 40 µg/mL coelenterazine (Biotium, Fremont, CA, USA) using an Orion L Microplate Luminometer (Berthold Detection Systems, Pforzheim, Germany) and an Infiniti M200 laser scanner (Tecan, Männedorf, Switzerland). Heat-inactivated serum was used as a negative control.

Staphylococcus aureus pRMC2, pRMC2:vanZ_Tei and pRMC2:vanZ_g, S. pneumoniae R6, S. pneumoniae R6, R6ΔvanZ, and R6ΔvanZ:vanZ cells were pregrown in Mueller Hinton medium (Oxoid/Thermo Fisher Scientific, Germany) to A_600nm = 0.4. S. aureus was pregrown in the presence of chloramphenicol and AnhTet. Cells were harvested by centrifugation and resuspended to A_600nm = 1 in 50 mM Tris–HCl buffer (pH = 7.4). Increasing amounts of Bodipy-Vancomycin (Thermo Fisher Scientific, Germany) or Fluorescent Teicoplanin (Vimberg et al., 2019 (link)) were added to 1 ml of resuspended cells. Cells were then incubated for 10 min at room temperature with the fluorescent antibiotics, harvested by centrifugation, washed two times with 50 mM Tris–HCl buffer (pH = 7.4), and finally resuspended in 100 μl of the same buffer. The fluorescence of 70 μl of resuspended cells was measured in automatic gain mode at Ex_490nm/Em_520nm in the case of fluorescent vancomycin or Ex_530nm/Em_580nm in the case of fluorescent teicoplanin in 96-well black plates (Thermo Fisher Scientific, Germany) by Tecan Infinite 200Pro. The experiment was repeated three times in duplicate.

After the treatment with BPF, cells were sedimented at 800× g, resuspended in phosphate-buffered saline (PBS) containing 10 μM DCFH-DA, incubated at 37 °C for 30 min in the dark, washed with PBS, and then seeded on 96-well black plates (Thermo Fisher Scientific, Waltham, MA, USA). The intensity of fluorescence was read at 480/530 nm (excitation/emission) using a microplate reader (Tecan Group Ltd., Männedorf, Switzerland).